Variant nucleic acid libraries for single domain antibodies

ABSTRACT

Provided herein are methods and compositions relating to variant nucleic acid libraries encoding for antibodies including single domain antibodies. Libraries generated using methods described herein have improved characteristics including improved binding affinity. Libraries described herein include variegated libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries generated when the nucleic acid libraries are translated. Further described herein are cell libraries expressing variegated nucleic acid libraries described herein.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/904,620 filed on Sep. 23, 2019; U.S. ProvisionalPatent Application No. 62/935,603 filed on Nov. 14, 2019; and U.S.Provisional Patent Application No. 62/945,761 filed on Dec. 9, 2019,each of which is incorporated by reference in its entirety.

BACKGROUND

Antibodies possess the capability to bind with high specificity andaffinity to biological targets. However, the design of therapeuticantibodies is challenging due to balancing of immunological effects withefficacy. Single domain antibodies such as VHH antibodies have severalbeneficial characteristics. Thus, there is a need to developcompositions and methods for generation of antibodies such as VHHantibodies for use in therapeutics.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are antibodies or antibody fragments comprising a CDRH1comprising an amino acid sequence at least about 90% identical to thatset forth in SEQ ID NOs: 152 or 155, a CDRH2 comprising an amino acidsequence at least about 90% identical to that set forth in SEQ ID NOs:153 or 156, and a CDRH3 comprising an amino acid sequence at least about90% identical to that set forth in SEQ ID NOs: 154 or 157. Furtherprovided herein are antibodies or antibody fragments, further comprisinga CDRL1 comprising an amino acid sequence at least about 90% identicalto that set forth in SEQ ID NOs: 158 or 161, a CDRL2 comprising an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNOs: 159 or 162, and a CDRL3 comprising an amino acid sequence at leastabout 90% identical to that set forth in SEQ ID NOs: 160 or 163.

Provided herein are methods of treating cancer comprising administeringthe antibody or antibody fragment described herein.

Provided herein are methods of treating a viral infection comprisingadministering the antibody or antibody fragment described herein.

Provided herein are nucleic acid libraries comprising: a plurality ofsequences comprising nucleic acids that when translated encode for anantibody or antibody fragment, wherein each sequence of the plurality ofsequences comprises a variant sequence encoding for a CDR1, CDR2, orCDR3 on a variable region of a heavy chain (VH) or a CDR1, CDR2, or CDR3on a variable region of a light chain (VL); wherein the librarycomprises at least 30,000 variant sequences; and wherein the antibody orantibody fragments bind to its antigen with a K_(D) of less than 100 nM.Further provided herein are nucleic acid libraries, wherein the antibodyis a single domain antibody. Further provided herein are nucleic acidlibraries, wherein the single domain antibody is a VHH antibody. Furtherprovided herein are nucleic acid libraries, wherein the antibody bindsto TIGIT. Further provided herein are nucleic acid libraries, whereinthe variable region of the heavy chain when translated comprises anamino acid sequence at least about 90% identical to that set forth inSEQ ID NOs: 84-100. Further provided herein are nucleic acid libraries,wherein the variable region of the light chain when translated comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NOs: 101-117. Further provided herein are nucleic acid libraries,wherein the CDR1, CDR2, or CDR3 on the variable region of the heavychain comprises an amino acid sequence at least about 90% identical tothat set forth in any one of SEQ ID NOs: 67-83 or 118-128. Furtherprovided herein are nucleic acid libraries, wherein the CDR1, CDR2, orCDR3 on the variable region of the light chain comprises an amino acidsequence at least about 90% identical to that set forth in any one ofSEQ ID NOs: 129-137. Further provided herein are nucleic acid libraries,wherein the antibody binds to CD47. Further provided herein are nucleicacid libraries, wherein the antibody binds to CD3 epsilon. Furtherprovided herein are nucleic acid libraries, wherein the variable regionof the heavy chain when translated comprises an amino acid sequence atleast about 90% identical to that set forth in SEQ ID NOs: 138-141.Further provided herein are nucleic acid libraries, wherein the variableregion of the light chain when translated comprises an amino acidsequence at least about 90% identical to that set forth in SEQ ID NOs:142-145. Further provided herein are nucleic acid libraries, wherein thenucleic acid library comprises at least 50,000 variant sequences.Further provided herein are nucleic acid libraries, wherein the nucleicacid library comprises at least 100,000 variant sequences. Furtherprovided herein are nucleic acid libraries, wherein the nucleic acidlibrary comprises at least 10⁵ non-identical nucleic acids. Furtherprovided herein are nucleic acid libraries, wherein the nucleic acidlibrary has a theoretical diversity of at least 10⁹ sequences.

Provided herein are nucleic acid libraries comprising: a plurality ofsequences comprising nucleic acids that when translated encode for asingle domain antibody, wherein each sequence of the plurality ofsequences comprises a variant sequence encoding for CDR1, CDR2, or CDR3on a variable region of a heavy chain (VH); wherein the librarycomprises at least 30,000 variant sequences; and wherein the antibody orantibody fragments bind to its antigen with a K_(D) of less than 100 nM.Further provided herein are nucleic acid libraries, wherein a length ofthe VH when translated is about 90 to about 100 amino acids. Furtherprovided herein are nucleic acid libraries, wherein a length of the VHwhen translated is about 100 to about 400 amino acids. Further providedherein are nucleic acid libraries, wherein a length of the VH is about270 to about 300 base pairs. Further provided herein are nucleic acidlibraries, wherein a length of the VH is about 300 to about 1200 basepairs. Further provided herein are nucleic acid libraries, wherein thesingle domain antibody is a VHH antibody. Further provided herein arenucleic acid libraries, wherein the antibody binds to TIGIT. Furtherprovided herein are nucleic acid libraries, wherein the CDR1, CDR2, orCDR3 on the variable region of the heavy chain comprises an amino acidsequence at least about 90% identical to that set forth in any one ofSEQ ID NOs: 67-83 or 118-128. Further provided herein are nucleic acidlibraries, wherein the variable region of the heavy chain whentranslated comprises an amino acid sequence at least about 90% identicalto that set forth in any one of SEQ ID NOs: 84-100. Further providedherein are nucleic acid libraries, wherein the CDR3 on the variableregion of the heavy chain comprises an amino acid sequence at leastabout 90% identical to that set forth in any one of SEQ ID NOs: 101-117.Further provided herein are nucleic acid libraries, wherein the antibodybinds to CD47. Further provided herein are nucleic acid libraries,wherein the antibody binds to CD3 epsilon. Further provided herein arenucleic acid libraries, wherein the variable region of the heavy chainwhen translated comprises an amino acid sequence at least about 90%identical to that set forth in SEQ ID NOs: 138-141. Further providedherein are nucleic acid libraries, wherein the nucleic acid librarycomprises at least 50,000 variant sequences. Further provided herein arenucleic acid libraries, wherein the nucleic acid library comprises atleast 100,000 variant sequences. Further provided herein are nucleicacid libraries, wherein the nucleic acid library comprises at least 10⁵non-identical nucleic acids. Further provided herein are nucleic acidlibraries, wherein the nucleic acid library has a theoretical diversityof at least 10⁹ sequences.

Provided herein are methods for generating a nucleic acid libraryencoding for a single domain antibody comprising: (a) providingpredetermined sequences encoding for: i. a first plurality ofpolynucleotides, wherein each polynucleotide of the first plurality ofpolynucleotides encodes for at least 1000 variant sequences encoding forCDR1 on a heavy chain; ii. a second plurality of polynucleotides,wherein each polynucleotide of the second plurality of polynucleotidesencodes for at least 1000 variant sequences encoding for CDR2 on a heavychain; iii. a third plurality of polynucleotides, wherein eachpolynucleotide of the third plurality of polynucleotides encodes for atleast 1000 variant sequences encoding for CDR3 on a heavy chain; and (b)mixing the first plurality of polynucleotides, the second plurality ofpolynucleotides, and the third plurality of polynucleotides to form thenucleic acid library of variant nucleic acids encoding for the singledomain antibody, and wherein at least about 70% of the variant nucleicacids encode for a single domain antibody that binds to its antigen witha K_(D) of less than 100 nM. Further provided herein are methods forgenerating a nucleic acid library, wherein the single domain antibodycomprises one heavy chain variable domain. Further provided herein aremethods for generating a nucleic acid library, wherein the single domainantibody is a VHH antibody. Further provided herein are methods forgenerating a nucleic acid library, wherein the single domain antibodybinds to TIGIT. Further provided herein are methods for generating anucleic acid library, wherein the single domain antibody comprises anamino acid sequence at least about 90% identical to that set forth inany one of SEQ ID NOs: 84-100 or 138-141. Further provided herein aremethods for generating a nucleic acid library, wherein the single domainantibody binds to CD47. Further provided herein are methods forgenerating a nucleic acid library, wherein the nucleic acid librarycomprises at least 50,000 variant sequences. Further provided herein aremethods for generating a nucleic acid library, wherein the nucleic acidlibrary comprises at least 100,000 variant sequences. Further providedherein are methods for generating a nucleic acid library, wherein thenucleic acid library comprises at least 10⁵ non-identical nucleic acids.Further provided herein are methods for generating a nucleic acidlibrary, wherein the nucleic acid library comprises at least onesequence encoding for the single domain antibody that binds to anantigen with a K_(D) of less than 75 nM. Further provided herein aremethods for generating a nucleic acid library, wherein the nucleic acidlibrary comprises at least one sequence encoding for the single domainantibody that binds to an antigen with a K_(D) of less than 50 nM.Further provided herein are methods for generating a nucleic acidlibrary, wherein the nucleic acid library comprises at least onesequence encoding for the single domain antibody that binds to anantigen with a K_(D) of less than 25 nM. Further provided herein aremethods for generating a nucleic acid library, wherein the nucleic acidlibrary comprises at least one sequence encoding for the single domainantibody that binds to an antigen with a K_(D) of less than 10 nM.Further provided herein are methods for generating a nucleic acidlibrary, wherein the nucleic acid library has a theoretical diversity ofat least 10⁹ sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a diagram of steps demonstrating an exemplary processworkflow for gene synthesis as disclosed herein.

FIG. 2 illustrates an example of a computer system.

FIG. 3 is a block diagram illustrating an architecture of a computersystem.

FIG. 4 is a diagram demonstrating a network configured to incorporate aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 5 is a block diagram of a multiprocessor computer system using ashared virtual address memory space.

FIGS. 6-7 depicts a graph of TIGIT affinity distribution for the VHHlibraries, depicting either the affinity threshold from 20 to 4000 (FIG.6) or the affinity threshold from 20 to 1000 (FIG. 7). Out of 140 VHHbinders, 51 variants were <100 nM and 90 variants were <200 nM.

FIG. 8 depicts graphs of CDR3 counts per length for ‘VHH library,’ ‘VHHshuffle’ library, and ‘VHH hShuffle library.’

FIG. 9 depicts a graph of a TIGIT:CD155 blockade assay for TIGIT VHH Fcbinders. Concentration of the TIGIT VHH Fc binders in nanomolar (nM) ison the x-axis and relative HRP signal is on the y-axis.

FIG. 10 depicts a graph of CD47 affinity distribution of the CD47 VHH Fcbinders. Affinity threshold (monovalent KD) is on the x-axis and countis on the y-axis for ‘VHH ratio’ library (horizontal bars), ‘VHHshuffle’ library (black bars), and ‘VHH hShuffle’ library (dotted bars).

FIG. 11 depicts a graph of CD47-SIRPalpha inhibition assay for CD47 VHHFc binders. Concentration of the CD47 VHH Fc binders in nanomolar (nM)is on the x-axis and relative HRP signal is on the y-axis.

FIGS. 12A-12B depict graphs of FACS analysis (FIG. 12A) and graphs of adose curve and specificity (FIG. 12B) of GLP1R-43-77.

FIGS. 13A-13B depict graphs of FACS analysis (FIG. 13A) and graphs of adose curve and cAMP activity (FIG. 13B) of CRTH2-41-51.

FIGS. 14A-14B depict graphs of a dose curve (FIG. 14A) and FACS analysis(FIG. 14B) of CRTH2-44-59.

FIGS. 15A-15E depict FACS analysis plots of cell binding as measured bymean fluorescence intensity (MFI) vs. 8-point titrations with CRTH2R IgGusing CRTH2-74, CRTH2-24, CRTH2-28, CRTH2-39, CRTH2-19, CRTH2-9,CRTH2-8, CRTH2-27, CRTH2-45, CRTH2-35, CRTH2-50, CRTH2-66, CRTH2-57,CRTH2-32, CRTH2-15, CRTH2-25, CRTH2-42, CRTH2-55, CRTH2-60, andCRTH2-70.

FIG. 16A depicts an example gated dot plot showing CRTH2-27 binding at100 nM.

FIG. 16B depicts an example APC histogram showing CRTH2-27 binding at100 nM.

FIG. 17A depicts binding analysis as in previous figures usingcomparator antibody gPCR-51.

FIG. 17B depicts binding analysis as in previous figures usingcomparator antibody gPCR-52.

FIGS. 18A-18B depict IgG binding curves with CRTH2-9, CRTH2-27,CRTH2-50, CRTH2-32, and CRTH2-42, which have functional effects in cAMPassays.

FIG. 19A depicts results of CRTH2R cAMP assays across all antibodiestested at 300, 100, and 33 nM.

FIG. 19B depicts results of CRTH2R cAMP assays across all antibodiestested at 33 nM.

FIG. 20 indicates the negative allosteric effect seen in five of theCRTH2R IgGs (CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, and CRTH2-42).

FIGS. 21A-21C depict control experiments of allosteric modulators,showing comparator antibody 52 is a positive allosteric modulator.

FIGS. 22A-22D depict activity of CRTH2R in β-arrestin recruitment assaysof CRTH2R IgGs.

FIG. 23 depicts a schema of libraries generated herein.

FIG. 24 depicts a schema of design of phage-displayed hyperimmunelibraries generated herein.

FIGS. 25A-25B depict heavy chain CDR length distribution of thehyperimmune libraries as assessed by next generation sequencing. FIG.25A depicts a graph of CDR3 counts per length. FIG. 25B depicts graphsof CDRH1, CDRH2, and CDRH3 lengths.

FIG. 26 depicts a schema of the workflow of selection of soluble proteintargets.

FIGS. 27A-27D depict graphs of data from hTIGIT ELISA after Round 3 andRound 4 of panning.

FIGS. 27E-27F depict schemas of CDRH3 length, yield, and affinity(K_(D)) for the hTIGIT immunoglobulins.

FIGS. 28A-28D depict graphs of data from human CD3 epsilon (hCD3) andcyno CD3 epsilon (cCD3) ELISA after Round 4 and Round 5 of panning.

FIGS. 28E-28L depict graphs of cross-reactive human CD3 epsilon (hCD3)and cyno CD3 epsilon (cCD3) immunoglobulins.

FIGS. 29A-29G depict graphs of titration of human CD3 on CD8+, CD3+, andCD3-T cells.

FIGS. 30A-30F depict graphs of binding affinity for the CRTH2Rimmunoglobulins CRTH2-48-03 (FIG. 30A), CRTH2-48-21 (FIG. 30B), andCRTH2-48-27 (FIG. 30C) and cAMP assays for CRTH2-48-03 (FIG. 30D),CRTH2-48-21 (FIG. 30E), and CRTH2-48-27 (FIG. 30F).

FIGS. 31A-31B depict graphs of a dose curve (FIG. 31A) and FACS analysis(FIG. 31B) of A2AR-90-007.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventionalmolecular biology techniques, which are within the skill of the art.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

Definitions

Throughout this disclosure, various embodiments are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

Unless specifically stated, as used herein, the term “nucleic acid”encompasses double- or triple-stranded nucleic acids, as well assingle-stranded molecules. In double- or triple-stranded nucleic acids,the nucleic acid strands need not be coextensive (i.e., adouble-stranded nucleic acid need not be double-stranded along theentire length of both strands). Nucleic acid sequences, when provided,are listed in the 5′ to 3′ direction, unless stated otherwise. Methodsdescribed herein provide for the generation of isolated nucleic acids.Methods described herein additionally provide for the generation ofisolated and purified nucleic acids. A “nucleic acid” as referred toherein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, or more bases in length. Moreover, providedherein are methods for the synthesis of any number ofpolypeptide-segments encoding nucleotide sequences, including sequencesencoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomalpeptide-synthetase (NRPS) modules and synthetic variants, polypeptidesegments of other modular proteins, such as antibodies, polypeptidesegments from other protein families, including non-coding DNA or RNA,such as regulatory sequences e.g. promoters, transcription factors,enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived frommicroRNA, or any functional or structural DNA or RNA unit of interest.The following are non-limiting examples of polynucleotides: coding ornon-coding regions of a gene or gene fragment, intergenic DNA, loci(locus) defined from linkage analysis, exons, introns, messenger RNA(mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA,ribozymes, complementary DNA (cDNA), which is a DNA representation ofmRNA, usually obtained by reverse transcription of messenger RNA (mRNA)or by amplification; DNA molecules produced synthetically or byamplification, genomic DNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. cDNAencoding for a gene or gene fragment referred herein may comprise atleast one region encoding for exon sequences without an interveningintron sequence in the genomic equivalent sequence.

Antibody Libraries

Provided herein are methods, compositions, and systems for generation ofantibodies. In some instances, the antibodies are single domainantibodies. Methods, compositions, and systems described herein for theoptimization of antibodies comprise a ratio-variant approach that mirrorthe natural diversity of antibody sequences. In some instances,libraries of optimized antibodies comprise variant antibody sequences.In some instances, the variant antibody sequences are designedcomprising variant CDR regions. In some instances, the variant antibodysequences comprising variant CDR regions are generated by shuffling thenatural CDR sequences in a llama, humanized, or chimeric framework. Insome instances, such libraries are synthesized, cloned into expressionvectors, and translation products (antibodies) evaluated for activity.In some instances, fragments of sequences are synthesized andsubsequently assembled. In some instances, expression vectors are usedto display and enrich desired antibodies, such as phage display. In someinstances, the phage vector is a Fab phagemid vector. Selectionpressures used during enrichment in some instances includes bindingaffinity, toxicity, immunological tolerance, stability, or other factor.Such expression vectors allow antibodies with specific properties to beselected (“panning”), and subsequent propagation or amplification ofsuch sequences enriches the library with these sequences. Panning roundscan be repeated any number of times, such as 1, 2, 3, 4, 5, 6, 7, ormore than 7 rounds. In some instances, each round of panning involves anumber of washes. In some instances, each round of panning involves atleast or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, ormore than 16 washes.

Described herein are methods and systems of in-silico library design.Libraries as described herein, in some instances, are designed based ona database comprising a variety of antibody sequences. In someinstances, the database comprises a plurality of variant antibodysequences against various targets. In some instances, the databasecomprises at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000,4500, 5000, or more than 5000 antibody sequences. An exemplary databaseis an iCAN database. In some instances, the database comprises naïve andmemory B-cell receptor sequences. In some instances, the naïve andmemory B-cell receptor sequences are human, mouse, or primate sequences.In some instances, the naïve and memory B-cell receptor sequences arehuman sequences. In some instances, the database is analyzed forposition specific variation. In some instances, antibodies describedherein comprise position specific variations in CDR regions. In someinstances, the CDR regions comprise multiple sites for variation.

Described herein are libraries comprising variation in a CDR region. Insome instances, the CDR is CDR1, CDR2, or CDR3 of a variable heavychain. In some instances, the CDR is CDR1, CDR2, or CDR3 of a variablelight chain. In some instances, the libraries comprise multiple variantsencoding for CDR1, CDR2, or CDR3. In some instances, the libraries asdescribed herein encode for at least 50, 100, 200, 300, 400, 500, 1000,1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than5000 CDR1 sequences. In some instances, the libraries as describedherein encode for at least 50, 100, 200, 300, 400, 500, 1000, 1200,1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000CDR2 sequences. In some instances, the libraries as described hereinencode for at least 50, 100, 200, 300, 400, 500, 1000, 1200, 1500, 1700,2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 CDR3sequences. In-silico antibodies libraries are in some instancessynthesized, assembled, and enriched for desired sequences.

Following synthesis of CDR1 variants, CDR2 variants, and CDR3 variants,in some instances, the CDR1 variants, the CDR2 variants, and the CDR3variants are shuffled to generate a diverse library. In some instances,the diversity of the libraries generated by methods described hereinhave a theoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, or more than 10¹⁸sequences. In some instances, the library has a final library diversityof at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, 10¹⁸, or more than 10¹⁸ sequences.

The germline sequences corresponding to a variant sequence may also bemodified to generate sequences in a library. For example, sequencesgenerated by methods described herein comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 mutations fromthe germline sequence. In some instances, sequences generated compriseno more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, orno more than 18 mutations from the germline sequence. In some instances,sequences generated comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, or about 18 mutations relative to the germlinesequence.

Antibody Libraries

Provided herein are libraries generated from methods described herein.Antibodies described herein result in improved functional activity,structural stability, expression, specificity, or a combination thereof.In some instances, the antibody is a single domain antibody. In someinstances, the single domain antibody comprises one heavy chain variabledomain. In some instances, the single domain antibody is a VHH antibody.

As used herein, the term antibody will be understood to include proteinshaving the characteristic two-armed, Y-shape of a typical antibodymolecule as well as one or more fragments of an antibody that retain theability to specifically bind to an antigen. Exemplary antibodiesinclude, but are not limited to, a monoclonal antibody, a polyclonalantibody, a bi-specific antibody, a multispecific antibody, a graftedantibody, a human antibody, a humanized antibody, a synthetic antibody,a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv)(including fragments in which the VL and VH are joined using recombinantmethods by a synthetic or natural linker that enables them to be made asa single protein chain in which the VL and VH regions pair to formmonovalent molecules, including single chain Fab and scFab), a singlechain antibody, a Fab fragment (including monovalent fragmentscomprising the VL, VH, CL, and CH1 domains), a F(ab′)2 fragment(including bivalent fragments comprising two Fab fragments linked by adisulfide bridge at the hinge region), a Fd fragment (includingfragments comprising the VH and CH1 fragment), a Fv fragment (includingfragments comprising the VL and VH domains of a single arm of anantibody), a single-domain antibody (dAb or sdAb) (including fragmentscomprising a VH domain), an isolated complementarity determining region(CDR), a diabody (including fragments comprising bivalent dimers such astwo VL and VH domains bound to each other and recognizing two differentantigens), a fragment comprised of only a single monomeric variabledomain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic(anti-Id) antibody, or ab antigen-binding fragments thereof. In someinstances, the libraries disclosed herein comprise nucleic acidsencoding for an antibody, wherein the antibody is a Fv antibody,including Fv antibodies comprised of the minimum antibody fragment whichcontains a complete antigen-recognition and antigen-binding site. Insome embodiments, the Fv antibody consists of a dimer of one heavy chainand one light chain variable domain in tight, non-covalent association,and the three hypervariable regions of each variable domain interact todefine an antigen-binding site on the surface of the VH-VL dimer. Insome embodiments, the six hypervariable regions confer antigen-bindingspecificity to the antibody. In some embodiments, a single variabledomain (or half of an Fv comprising only three hypervariable regionsspecific for an antigen, including single domain antibodies isolatedfrom camelid animals comprising one heavy chain variable domain such asVHH antibodies or nanobodies) has the ability to recognize and bindantigen. In some instances, the libraries disclosed herein comprisenucleic acids encoding for an antibody, wherein the antibody is asingle-chain Fv or scFv, including antibody fragments comprising a VH, aVL, or both a VH and VL domain, wherein both domains are present in asingle polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the VH and VL domainsallowing the scFv to form the desired structure for antigen binding. Insome instances, a scFv is linked to the Fc fragment or a VHH is linkedto the Fc fragment (including minibodies). In some instances, theantibody comprises immunoglobulin molecules and immunologically activefragments of immunoglobulin molecules, e.g., molecules that contain anantigen binding site. Immunoglobulin molecules are of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG4, IgA 1 and IgA 2) or subclass.

In some embodiments, libraries comprise immunoglobulins that are adaptedto the species of an intended therapeutic target. Generally, thesemethods include “mammalization” and comprises methods for transferringdonor antigen-binding information to a less immunogenic mammal antibodyacceptor to generate useful therapeutic treatments. In some instances,the mammal is mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee,baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, andhuman. In some instances, provided herein are libraries and methods forfelinization and caninization of antibodies.

“Humanized” forms of non-human antibodies can be chimeric antibodiesthat contain minimal sequence derived from the non-human antibody. Ahumanized antibody is generally a human antibody (recipient antibody) inwhich residues from one or more CDRs are replaced by residues from oneor more CDRs of a non-human antibody (donor antibody). The donorantibody can be any suitable non-human antibody, such as a mouse, rat,rabbit, chicken, or non-human primate antibody having a desiredspecificity, affinity, or biological effect. In some instances, selectedframework region residues of the recipient antibody are replaced by thecorresponding framework region residues from the donor antibody.Humanized antibodies may also comprise residues that are not found ineither the recipient antibody or the donor antibody. In some instances,these modifications are made to further refine antibody performance.

“Caninization” can comprise a method for transferring non-canineantigen-binding information from a donor antibody to a less immunogeniccanine antibody acceptor to generate treatments useful as therapeuticsin dogs. In some instances, caninized forms of non-canine antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-canine antibodies. In some instances, caninizedantibodies are canine antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-canine species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non-human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the canine antibody are replaced by corresponding non-canineFR residues. In some instances, caninized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The caninized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a canineantibody.

“Felinization” can comprise a method for transferring non-felineantigen-binding information from a donor antibody to a less immunogenicfeline antibody acceptor to generate treatments useful as therapeuticsin cats. In some instances, felinized forms of non-feline antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-feline antibodies. In some instances, felinizedantibodies are feline antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-feline species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non-human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the feline antibody are replaced by corresponding non-felineFR residues. In some instances, felinized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The felinized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a felinizeantibody.

Methods as described herein may be used for generation of librariesencoding a non-immunoglobulin. In some instances, the libraries compriseantibody mimetics. Exemplary antibody mimetics include, but are notlimited to, anticalins, affilins, affibody molecules, affimers,affitins, alphabodies, avimers, atrimers, DARPins, fynomers, Kunitzdomain-based proteins, monobodies, anticalins, knottins, armadillorepeat protein-based proteins, and bicyclic peptides.

Libraries described herein comprising nucleic acids encoding for anantibody comprise variations in at least one region of the antibody.Exemplary regions of the antibody for variation include, but are notlimited to, a complementarity-determining region (CDR), a variabledomain, or a constant domain. In some instances, the CDR is CDR1, CDR2,or CDR3. In some instances, the CDR is a heavy domain including, but notlimited to, CDRH1, CDRH2, and CDRH3. In some instances, the CDR is alight domain including, but not limited to, CDRL1, CDRL2, and CDRL3. Insome instances, the variable domain is variable domain, light chain (VL)or variable domain, heavy chain (VH). In some instances, the CDR1, CDR2,or CDR3 is of a variable domain, light chain (VL). CDR1, CDR2, or CDR3of a variable domain, light chain (VL) can be referred to as CDRL1,CDRL2, or CDRL3, respectively. CDR1, CDR2, or CDR3 of a variable domain,heavy chain (VH) can be referred to as CDRH1, CDRH2, or CDRH3,respectively. In some instances, the VL domain comprises kappa or lambdachains. In some instances, the constant domain is constant domain, lightchain (CL) or constant domain, heavy chain (CH).

Provided herein are libraries comprising nucleic acids encoding for anantibody comprising variation in at least one region of the antibody,wherein the region is the CDR region. In some instances, the antibody isa single domain antibody comprising one heavy chain variable domain suchas a VHH antibody. In some instances, the VHH antibody comprisesvariation in one or more CDR regions. In some instances, the VHHlibraries described herein comprise at least or about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400,2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3.For example, the libraries comprise at least 2000 sequences of a CDR1,at least 1200 sequences for CDR2, and at least 1600 sequences for CDR3.In some instances, each sequence is non-identical.

Libraries as described herein may comprise varying lengths of a CDRH1,CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, or combinations thereof of aminoacids when translated. In some instances, the length of the CDRH1,CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, or combinations thereof of aminoacids when translated is at least or about 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or more than 30 amino acids.

Libraries comprising nucleic acids encoding for antibodies havingvariant CDR sequences as described herein comprise various lengths ofamino acids when translated. In some instances, the length of each ofthe amino acid fragments or average length of the amino acid synthesizedmay be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, or more than 150 amino acids. In some instances, the length of theamino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105,70 to 100, or 75 to 95 amino acids. In some instances, the length of theamino acid is about 22 amino acids to about 75 amino acids. In someinstances, the antibodies comprise at least or about 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000amino acids. In some instances, the library is a VHH library. In someinstances, the library is an antibody library.

Libraries as described herein encoding for a VHH antibody comprisevariant CDR sequences that are shuffled to generate a library with atheoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, or more than 10¹⁸ sequences.In some instances, the library has a final library diversity of at leastor about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷,10¹⁸, or more than 10¹⁸ sequences.

Libraries as described herein encoding for an antibody or immunoglobulincomprise variant CDR sequences that are shuffled to generate a librarywith a theoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, or more than 10¹⁸sequences. In some instances, the library has a final library diversityof at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, 10¹⁸, or more than 10¹⁸ sequences.

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding an antibody or immunoglobulin, wherein eachnucleic acid encodes for a predetermined variant of at least onepredetermined reference nucleic acid sequence. In some cases, thepredetermined reference sequence is a nucleic acid sequence encoding fora protein, and the variant library comprises sequences encoding forvariation of at least a single codon such that a plurality of differentvariants of a single residue in the subsequent protein encoded by thesynthesized nucleic acid are generated by standard translationprocesses. In some instances, the antibody library comprises variednucleic acids collectively encoding variations at multiple positions. Insome instances, the variant library comprises sequences encoding forvariation of at least a single codon of a CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, CDRL3, VL, or VH domain. In some instances, the variant librarycomprises sequences encoding for variation of multiple codons of aCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In someinstances, the variant library comprises sequences encoding forvariation of multiple codons of framework element 1 (FW1), frameworkelement 2 (FW2), framework element 3 (FW3), or framework element 4(FW4). An exemplary number of codons for variation include, but are notlimited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275,300, or more than 300 codons.

In some instances, the at least one region of the antibody for variationis from heavy chain V-gene family, heavy chain D-gene family, heavychain J-gene family, light chain V-gene family, or light chain J-genefamily. In some instances, the light chain V-gene family comprisesimmunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL).

Provided herein are libraries comprising nucleic acids encoding forantibodies, wherein the libraries are synthesized with various numbersof fragments. In some instances, the fragments comprise the CDRH1,CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances,the fragments comprise framework element 1 (FW1), framework element 2(FW2), framework element 3 (FW3), or framework element 4 (FW4). In someinstances, the antibody libraries are synthesized with at least or about2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5fragments. The length of each of the nucleic acid fragments or averagelength of the nucleic acids synthesized may be at least or about 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In someinstances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to375, or 300 to 350 base pairs.

Libraries comprising nucleic acids encoding for antibodies orimmunoglobulins as described herein comprise various lengths of aminoacids when translated. In some instances, the length of each of theamino acid fragments or average length of the amino acid synthesized maybe at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,or more than 150 amino acids. In some instances, the length of the aminoacid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to100, or 75 to 95 amino acids. In some instances, the length of the aminoacid is about 22 amino acids to about 75 amino acids. In some instances,the antibodies comprise at least or about 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 aminoacids.

A number of variant sequences for the at least one region of theantibody for variation are de novo synthesized using methods asdescribed herein. In some instances, a number of variant sequences is denovo synthesized for CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH,or combinations thereof. In some instances, a number of variantsequences is de novo synthesized for framework element 1 (FW1),framework element 2 (FW2), framework element 3 (FW3), or frameworkelement 4 (FW4). The number of variant sequences may be at least orabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, or more than 500 sequences. In some instances, thenumber of variant sequences is at least or about 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000sequences. In some instances, the number of variant sequences is about10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325sequences.

Variant sequences for the at least one region of the antibody, in someinstances, vary in length or sequence. In some instances, the at leastone region that is de novo synthesized is for CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances,the at least one region that is de novo synthesized is for frameworkelement 1 (FW1), framework element 2 (FW2), framework element 3 (FW3),or framework element 4 (FW4). In some instances, the variant sequencecomprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, or more than 50 variant nucleotides or amino acidsas compared to wild-type. In some instances, the variant sequencecomprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, or 50 additional nucleotides or amino acids as comparedto wild-type. In some instances, the variant sequence comprises at leastor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or50 less nucleotides or amino acids as compared to wild-type. In someinstances, the libraries comprise at least or about 10¹, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more than 10¹⁰ variants.

Following synthesis of antibody libraries, antibody libraries may beused for screening and analysis. For example, antibody libraries areassayed for library displayability and panning. In some instances,displayability is assayed using a selectable tag. Exemplary tagsinclude, but are not limited to, a radioactive label, a fluorescentlabel, an enzyme, a chemiluminescent tag, a colorimetric tag, anaffinity tag or other labels or tags that are known in the art. In someinstances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA),or FLAG. For example, as seen in FIG. 2B. In some instances, antibodylibraries are assayed by sequencing using various methods including, butnot limited to, single-molecule real-time (SMRT) sequencing, Polonysequencing, sequencing by ligation, reversible terminator sequencing,proton detection sequencing, ion semiconductor sequencing, nanoporesequencing, electronic sequencing, pyrosequencing, Maxam-Gilbertsequencing, chain termination (e.g., Sanger) sequencing, +S sequencing,or sequencing by synthesis. In some instances, antibody libraries aredisplayed on the surface of a cell or phage. In some instances, antibodylibraries are enriched for sequences with a desired activity using phagedisplay.

In some instances, the antibody libraries are assayed for functionalactivity, structural stability (e.g., thermal stable or pH stable),expression, specificity, or a combination thereof. In some instances,the antibody libraries are assayed for antibody capable of folding. Insome instances, a region of the antibody is assayed for functionalactivity, structural stability, expression, specificity, folding, or acombination thereof. For example, a VH region or VL region is assayedfor functional activity, structural stability, expression, specificity,folding, or a combination thereof.

Antibodies or IgGs generated by methods as described herein compriseimproved binding affinity. In some instances, the antibody comprises abinding affinity (e.g., K_(D)) of less than 1 nM, less than 1.2 nM, lessthan 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than30 nM. In some instances, the antibody comprises a K_(D) of less than400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than200 nM, less than 150 nm, less than 100 nM, less than 50 nM, less than25 nM, less than 15 nM, or less than 10 nM. In some instances, theantibody comprises a K_(D) of less than 1 nM. In some instances, theantibody comprises a K_(D) of less than 1.2 nM. In some instances, theantibody comprises a K_(D) of less than 2 nM. In some instances, theantibody comprises a K_(D) of less than 5 nM. In some instances, theantibody comprises a K_(D) of less than 10 nM. In some instances, theantibody comprises a K_(D) of less than 13.5 nM. In some instances, theantibody comprises a K_(D) of less than 15 nM. In some instances, theantibody comprises a K_(D) of less than 20 nM. In some instances, theantibody comprises a K_(D) of less than 25 nM. In some instances, theantibody comprises a K_(D) of less than 30 nM.

In some instances, the affinity of antibodies or IgGs generated bymethods as described herein is at least or about 1.5×, 2.0×, 5×, 10×,20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, or more than 200×improved binding affinity as compared to a comparator antibody. In someinstances, the affinity of antibodies or IgGs generated by methods asdescribed herein is at least or about 1.5×, 2.0×, 5×, 10×, 20×, 30×,40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, or more than 200× improvedfunction as compared to a comparator antibody. In some instances, thecomparator antibody is an antibody with similar structure, sequence, orantigen target.

Methods as described herein, in some instances, result in increasedyield of antibodies or IgGs. In some instances, the yield is at least orabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, ormore than 80 micrograms (ug). In some instances, the yield is in a rangeof about 5 to about 80, about 10 to about 75, about 15 to about 60,about 20 to about 50, or about 30 to about 40 micrograms (ug).

Expression Systems

Provided herein are libraries comprising nucleic acids encoding forantibody comprising binding domains, wherein the libraries have improvedspecificity, stability, expression, folding, or downstream activity. Insome instances, libraries described herein are used for screening andanalysis.

Provided herein are libraries comprising nucleic acids encoding forantibody comprising binding domains, wherein the nucleic acid librariesare used for screening and analysis. In some instances, screening andanalysis comprises in vitro, in vivo, or ex vivo assays. Cells forscreening include primary cells taken from living subjects or celllines. Cells may be from prokaryotes (e.g., bacteria and fungi) oreukaryotes (e.g., animals and plants). Exemplary animal cells include,without limitation, those from a mouse, rabbit, primate, and insect. Insome instances, cells for screening include a cell line including, butnot limited to, Chinese Hamster Ovary (CHO) cell line, human embryonickidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In someinstances, nucleic acid libraries described herein may also be deliveredto a multicellular organism. Exemplary multicellular organisms include,without limitation, a plant, a mouse, rabbit, primate, and insect.

Nucleic acid libraries described herein may be screened for variouspharmacological or pharmacokinetic properties. In some instances, thelibraries are screened using in vitro assays, in vivo assays, or ex vivoassays. For example, in vitro pharmacological or pharmacokineticproperties that are screened include, but are not limited to, bindingaffinity, binding specificity, and binding avidity. Exemplary in vivopharmacological or pharmacokinetic properties of libraries describedherein that are screened include, but are not limited to, therapeuticefficacy, activity, preclinical toxicity properties, clinical efficacyproperties, clinical toxicity properties, immunogenicity, potency, andclinical safety properties.

Provided herein are nucleic acid libraries, wherein the nucleic acidlibraries may be expressed in a vector. Expression vectors for insertingnucleic acid libraries disclosed herein may comprise eukaryotic orprokaryotic expression vectors. Exemplary expression vectors include,without limitation, mammalian expression vectors:pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG,pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 Vector,pEF1a-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro,pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors:pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plantexpression vectors: pRI 101-AN DNA and pCambia2301; and yeast expressionvectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1/V5-His A andpDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in avector to generate a construct comprising an antibody. In someinstances, a size of the construct varies. In some instances, theconstruct comprises at least or about 500, 600, 700, 800, 900, 1000,1100, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000,3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 6000, 7000,8000, 9000, 10000, or more than 10000 bases. In some instances, a theconstruct comprises a range of about 300 to 1,000, 300 to 2,000, 300 to3,000, 300 to 4,000, 300 to 5,000, 300 to 6,000, 300 to 7,000, 300 to8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2,000, 1,000 to 3,000,1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1,000 to 7,000, 1,000 to8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000,2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000,3,000 to 7,000, 3,000 to 8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000to 5,000, 4,000 to 6,000, 4,000 to 7,000, 4,000 to 8,000, 4,000 to9,000, 4,000 to 10,000, 5,000 to 6,000, 5,000 to 7,000, 5,000 to 8,000,5,000 to 9,000, 5,000 to 10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000to 9,000, 6,000 to 10,000, 7,000 to 8,000, 7,000 to 9,000, 7,000 to10,000, 8,000 to 9,000, 8,000 to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding forantibodies, wherein the nucleic acid libraries are expressed in a cell.In some instances, the libraries are synthesized to express a reportergene. Exemplary reporter genes include, but are not limited to,acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), betagalactosidase (LacZ), beta glucoronidase (GUS), chloramphenicolacetyltransferase (CAT), green fluorescent protein (GFP), redfluorescent protein (RFP), yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), cerulean fluorescent protein, citrinefluorescent protein, orange fluorescent protein, cherry fluorescentprotein, turquoise fluorescent protein, blue fluorescent protein,horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS),octopine synthase (OCS), luciferase, and derivatives thereof. Methods todetermine modulation of a reporter gene are well known in the art, andinclude, but are not limited to, fluorometric methods (e.g. fluorescencespectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescencemicroscopy), and antibiotic resistance determination.

Diseases and Disorders

Provided herein are libraries comprising nucleic acids encoding forantibodies or immunoglobulins including VHH antibodies that may havetherapeutic effects. In some instances, the antibodies or immunoglobulinresult in protein when translated that is used to treat a disease ordisorder in a subject. Exemplary diseases include, but are not limitedto, cancer, inflammatory diseases or disorders, a metabolic disease ordisorder, a cardiovascular disease or disorder, a respiratory disease ordisorder, pain, a digestive disease or disorder, a reproductive diseaseor disorder, an endocrine disease or disorder, or a neurological diseaseor disorder. In some instances, the cancer is a solid cancer or ahematologic cancer. In some instances, the subject is a mammal. In someinstances, the subject is a mouse, rabbit, dog, or human. Subjectstreated by methods described herein may be infants, adults, or children.Pharmaceutical compositions comprising antibodies or antibody fragmentsas described herein may be administered intravenously or subcutaneously.

In some instances, the disease or disorder is associated with TIGITdysfunction. In some instances, the disease or disorder is associatedwith aberrant signaling via TIGIT. In some instances, the disease ordisorder is associated with CD3 dysfunction. In some instances, thedisease or disorder is associated with aberrant signaling via CD3. Insome instances, the disease or disorder is cancer. In some instances,the disease or disorder is a viral infection.

Protein Targets

Provided herein are libraries comprising nucleic acids encoding forantibodies or immunoglobulins including VHH antibodies that may bedesigned for various protein targets. In some instances, the protein isan ion channel, G protein-coupled receptor, tyrosine kinase receptor, animmune receptor, a membrane protein, or combinations thereof. In someinstances, the protein is a receptor. In some instances, the protein isGlucagon-like peptide 1 (GLP1) receptor. In some instances, the proteinis Prostaglandin D2 receptor 2 (DP2 or CRTH2) receptor. In someinstances, the protein is an adenosine A2A receptor. In some instances,the protein is T cell immunoreceptor with Ig and ITIM domains (TIGIT).In some instances, the protein is Cluster of Differentiation 47 (CD47).In some instances, the protein is Cluster of Differentiation 3 epsilon(CD3E).

Provided herein are antibodies or immunoglobulins, wherein the antibodyor immunoglobulin comprises a sequence at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 1-151. In some instances, theantibody or immunoglobulin sequence comprises at least or about 95%sequence identity to any one of SEQ ID NOs: 1-151. In some instances,the antibody or immunoglobulin sequence comprises at least or about 97%sequence identity to any one of SEQ ID NOs: 1-151. In some instances,the antibody or immunoglobulin sequence comprises at least or about 99%sequence identity to any one of SEQ ID NOs: 1-151. In some instances,the antibody or immunoglobulin sequence comprises at least or about 100%sequence identity to any one SEQ ID NOs: 1-151. In some instances, theantibody or immunoglobulin sequence comprises at least a portion havingat least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids ofany one of SEQ ID NOs: 1-151.

In some embodiments, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising a sequence as setforth in Table 1A, Table 14B, Table 17, and Table 20. In someembodiments, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to any one of SEQ ID NOs: 46-83, 118-137, or 152-163.In some instances, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about95% homology to any one of SEQ ID NOs: 46-83, 118-137, or 152-163. Insome instances, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about97% homology to any one of SEQ ID NOs: 46-83, 118-137, or 152-163. Insome instances, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about99% homology to any one of SEQ ID NOs: 46-83, 118-137, or 152-163. Insome instances, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about100% homology to any one of SEQ ID NOs: 46-83, 118-137, or 152-163. Insome instances, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of any one of SEQ ID NOs: 46-83, 118-137, or152-163.

TABLE 1A Construct Amino  SEQ ID Description Acid Sequence NOIGHV1-69 CDRH1 GGTFSSYA 152 IGHV1-69 CDRH2 IIPIFGTA 153 IGHV1-69 CDRH3CARNNNNNNNNNFDYW 154 IGHV3-23 CDRH1 GFTFSSYA 155 IGHV3-23 CDRH2 ISGSGGST156 IGHV3-23 CDRH3 CAKNNNNNNNNNFDYW 157 IGKV1-39 CDRL1 QSISSY 158IGKV1-39 CDRL2 AAS 159 IGKV1-39 CDRL3 CQQSYSTPNTF 160 IGKV3-20 CDRL1QSVSSSY 161 IGKV3-20 CDRL2 GAS 162 IGKV3-20 CDRL3 CQQYGSSPNTF 163

In some embodiments, the antibody or immunoglobulin sequence comprises aCDR1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 118-120, 129-131, 152, 155, 158, or 161. In some instances,the antibody or immunoglobulin sequence comprises CDR1 comprising atleast or about 95% homology of any one of SEQ ID NOs: 118-120, 129-131,152, 155, 158, or 161. In some instances, the antibody or immunoglobulinsequence comprises CDR1 comprising at least or about 97% homology to anyone of SEQ ID NOs: 118-120, 129-131, 152, 155, 158, or 161. In someinstances, the antibody or immunoglobulin sequence comprises CDR1comprising at least or about 99% homology to any one of SEQ ID NOs:118-120, 152, 155, 158, or 161. In some instances, the antibody orimmunoglobulin sequence comprises CDR1 comprising at least or about 100%homology to any one of SEQ ID NOs: 118-120, 129-131, 152, 155, 158, or161. In some instances, the antibody or immunoglobulin sequencecomprises CDR1 comprising at least a portion having at least or about 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of any oneof SEQ ID NOs: 118-120, 129-131, 152, 155, 158, or 161.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDR2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 121-123, 132-134, 153, 156, 159, or 162. In some instances,the antibody or immunoglobulin sequence comprises CDR2 comprising atleast or about 95% homology to any one of SEQ ID NOs: 121-123, 132-134,153, 156, 159, or 162. In some instances, the antibody or immunoglobulinsequence comprises CDR2 comprising at least or about 97% homology to anyone of SEQ ID NOs: 121-123, 132-134, 153, 156, 159, or 162. In someinstances, the antibody or immunoglobulin sequence comprises CDR2comprising at least or about 99% homology to any one of SEQ ID NOs:121-123, 132-134, 153, 156, 159, or 162. In some instances, the antibodyor immunoglobulin sequence comprises CDR2 comprising at least or about100% homology to any one of SEQ ID NOs: 121-123, 132-134, 153, 156, 159,or 162. In some instances, the antibody or immunoglobulin sequencecomprises CDR2 comprising at least a portion having at least or about 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of any oneof SEQ ID NOs: 121-123, 132-134, 153, 156, 159, or 162.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDR3 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 46-83, 124-128, 154, 157, 160, or 163. In some instances,the antibody or immunoglobulin sequence comprises CDR3 comprising atleast or about 95% homology to any one of SEQ ID NOs: 46-83, 124-128,125-137, 154, 157, 160, or 163. In some instances, the antibody orimmunoglobulin sequence comprises CDR3 comprising at least or about 97%homology to any one of SEQ ID NOs: 46-83, 124-128, 125-137, 124-128,154, 157, 160, or 163. In some instances, the antibody or immunoglobulinsequence comprises CDR3 comprising at least or about 99% homology to anyone of SEQ ID NOs: 46-83, 124-128, 125-137, 124-128, 154, 157, 160, or163. In some instances, the antibody or immunoglobulin sequencecomprises CDR3 comprising at least or about 100% homology to any one ofSEQ ID NOs: 46-83, 124-128, 125-137, 124-128, 154, 157, 160, or 163. Insome instances, the antibody or immunoglobulin sequence comprises CDR3comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of any one of SEQ ID NOs:46-83, 124-128, 125-137, 124-128, 154, 157, 160, or 163.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 152; a CDRH2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 153; and a CDRH3 comprising at leastor about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to any one of SEQ ID NOs: 154. In someinstances, the antibody or immunoglobulin sequence comprises CDRH1comprising at least or about 95%, 97%, 99%, or 100% homology to any oneof SEQ ID NOs: 152; a CDRH2 comprising at least or about 95%, 97%, 99%,or 100% homology to any one of SEQ ID NOs: 153; and a CDRH3 comprisingat least or about 95%, 97%, 99%, or 100% homology to any one of SEQ IDNOs: 154. In some instances, the antibody or immunoglobulin sequencecomprises CDRH1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 152; a CDRH2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 153; and a CDRH3 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 154.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:155; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 156; and a CDRH3 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 157. In some instances, the antibody or immunoglobulinsequence comprises CDRH1 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 155; a CDRH2 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 156; and a CDRH3comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 157. In some instances, the antibody or immunoglobulin sequencecomprises CDRH1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 155; a CDRH2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 156; and a CDRH3 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 157.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRL1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:158; a CDRL2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 159; and a CDRL3 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 160. In some instances, the antibody or immunoglobulinsequence comprises CDRL1 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 158; a CDRL2 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 159; and a CDRL3comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 160. In some instances, the antibody or immunoglobulin sequencecomprises CDRL1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 158; a CDRL2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 159; and a CDRL3 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 160.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRL1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:161; a CDRL2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 162; and a CDRL3 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 163. In some instances, the antibody or immunoglobulinsequence comprises CDRL1 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 161; a CDRL2 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 162; and a CDRL3comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 163. In some instances, the antibody or immunoglobulin sequencecomprises CDRL1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 161; a CDRL2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 162; and a CDRL3 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 163.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:152; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 153; a CDRH3 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 154, a CDRL1 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 158; a CDRL2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 159; and a CDRL3 comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 160. In some instances, the antibody orimmunoglobulin sequence comprises CDRH1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 152; a CDRH2 comprising atleast or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 153; aCDRH3 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 154; a CDRL1 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 158; a CDRL2 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 159; and a CDRL3comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 160. In some instances, the antibody or immunoglobulin sequencecomprises CDRH1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 152; a CDRH2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 153; a CDRH3 comprising at least a portion having at leastor about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 aminoacids of SEQ ID NO: 154; a CDRL1 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 158; a CDRL2 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 159; and a CDRL3 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 160.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:152; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 153; a CDRH3 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 154, a CDRL1 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 161; a CDRL2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 162; and a CDRL3 comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 163. In some instances, the antibody orimmunoglobulin sequence comprises CDRH1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 152; a CDRH2 comprising atleast or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 153; aCDRH3 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 154; a CDRL1 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 161; a CDRL2 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 162; and a CDRL3comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 163. In some instances, the antibody or immunoglobulin sequencecomprises CDRH1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 152; a CDRH2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 153; a CDRH3 comprising at least a portion having at leastor about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 aminoacids of SEQ ID NO: 154; a CDRL1 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 161; a CDRL2 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 162; and a CDRL3 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 163.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:155; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 156; a CDRH3 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 157, a CDRL1 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 158; a CDRL2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 159; and a CDRL3 comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 160. In some instances, the antibody orimmunoglobulin sequence comprises CDRH1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 155; a CDRH2 comprising atleast or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 156; aCDRH3 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 157; a CDRL1 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 158; a CDRL2 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 159; and a CDRL3comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 160. In some instances, the antibody or immunoglobulin sequencecomprises CDRH1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 155; a CDRH2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 156; a CDRH3 comprising at least a portion having at leastor about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 aminoacids of SEQ ID NO: 157; a CDRL1 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 158; a CDRL2 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 159; and a CDRL3 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 160.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:155; a CDRH2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 156; a CDRH3 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 157, a CDRL1 comprising at least or about 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NO: 161; a CDRL2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 162; and a CDRL3 comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 163. In some instances, the antibody orimmunoglobulin sequence comprises CDRH1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 155; a CDRH2 comprising atleast or about 95%, 97%, 99%, or 100% homology to SEQ ID NO: 156; aCDRH3 comprising at least or about 95%, 97%, 99%, or 100% homology toSEQ ID NO: 157; a CDRL1 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 161; a CDRL2 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 162; and a CDRL3comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 163. In some instances, the antibody or immunoglobulin sequencecomprises CDRH1 comprising at least a portion having at least or about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of SEQID NO: 155; a CDRH2 comprising at least a portion having at least orabout 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acidsof SEQ ID NO: 156; a CDRH3 comprising at least a portion having at leastor about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 aminoacids of SEQ ID NO: 157; a CDRL1 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of SEQ ID NO: 161; a CDRL2 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 162; and a CDRL3 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 163.

Described herein, in some embodiments, are antibodies or immunoglobulinsthat bind to the CRTH2R. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ IDNOs: 1-23 or 126-148. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 95% sequence identity to any one of SEQ IDNOs: 1-23 or 126-148. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 97% sequence identity to any one of SEQ IDNOs: 1-23 or 126-148. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 99% sequence identity to any one of SEQ IDNOs: 1-23 or 126-148. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 100% sequence identity to any one of SEQ IDNOs: 1-23 or 126-148. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least a portion having at least or about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, or more than 400 amino acids of SEQ ID NOs: 1-23 or 126-148.

In some instances, the CRTH2R antibody or immunoglobulin sequencecomprises a light chain variable domain comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to any one of SEQ ID NOs: 24-45 or 149-151. In someinstances, the CRTH2R antibody or immunoglobulin sequence comprises alight chain variable domain comprising at least or about 95% sequenceidentity to any one of SEQ ID NOs: 24-45 or 149-151. In some instances,the CRTH2R antibody or immunoglobulin sequence comprises a light chainvariable domain comprising at least or about 97% sequence identity toany one of SEQ ID NOs: 24-45 or 149-151. In some instances, the CRTH2Rantibody or immunoglobulin sequence comprises a light chain variabledomain comprising at least or about 99% sequence identity to any one ofSEQ ID NOs: 24-45 or 149-151. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a light chain variable domaincomprising at least or about 100% sequence identity to any one of SEQ IDNOs: 24-45 or 149-151. In some instances, the CRTH2R antibody orimmunoglobulin sequence comprises a light chain variable domaincomprising at least a portion having at least or about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, or more than 400 amino acids of SEQ ID NOs: 24-45 or 149-151.

Provided herein are antibodies or immunoglobulins for various proteintargets. In some instances, the protein is TIGIT. Described herein, insome embodiments, are antibodies or immunoglobulins that bind to theTIGIT. In some instances, the TIGIT antibody or immunoglobulin sequencecomprises a heavy chain variable domain comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to any one of SEQ ID NOs: 84-100. In some instances,the TIGIT antibody or immunoglobulin sequence comprises a heavy chainvariable domain comprising at least or about 95% sequence identity toany one of SEQ ID NOs: 84-100. In some instances, the TIGIT antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 97% sequence identity to any one of SEQ IDNOs: 84-100. In some instances, the TIGIT antibody or immunoglobulinsequence comprises a heavy chain variable domain comprising at least orabout 99% sequence identity to any one of SEQ ID NOs: 84-100. In someinstances, the TIGIT antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least or about 100% sequenceidentity to any one of SEQ ID NOs: 84-100. In some instances, the TIGITantibody or immunoglobulin sequence comprises a heavy chain variabledomain comprising at least a portion having at least or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, or more than 400 amino acids of any one of SEQ ID NOs:84-100.

In some instances, the TIGIT antibody or immunoglobulin sequencecomprises a light chain variable domain comprising at least or about70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to any one of SEQ ID NOs: 101-117. In some instances,the TIGIT antibody or immunoglobulin sequence comprises a light chainvariable domain comprising at least or about 95% sequence identity toany one of SEQ ID NOs: 101-117. In some instances, the TIGIT antibody orimmunoglobulin sequence comprises a light chain variable domaincomprising at least or about 97% sequence identity to any one of SEQ IDNOs: 101-117. In some instances, the TIGIT antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least orabout 99% sequence identity to any one of SEQ ID NOs: 101-117. In someinstances, the TIGIT antibody or immunoglobulin sequence comprises alight chain variable domain comprising at least or about 100% sequenceidentity to any one of SEQ ID NOs: 101-117. In some instances, the TIGITantibody or immunoglobulin sequence comprises a light chain variabledomain comprising at least a portion having at least or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, or more than 400 amino acids of any one of SEQ ID NOs:101-117.

In some instances, the protein is CD3 epsilon. Described herein, in someembodiments, are antibodies or immunoglobulins that bind to the CD3. Insome instances, the CD3 antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 138-141. In some instances, the CD3antibody or immunoglobulin sequence comprises a heavy chain variabledomain comprising at least or about 95% sequence identity to any one ofSEQ ID NOs: 138-141. In some instances, the CD3 antibody orimmunoglobulin sequence comprises a heavy chain variable domaincomprising at least or about 97% sequence identity to any one of SEQ IDNOs: 138-141. In some instances, the CD3 antibody or immunoglobulinsequence comprises a heavy chain variable domain comprising at least orabout 99% sequence identity to any one of SEQ ID NOs: 138-141. In someinstances, the CD3 antibody or immunoglobulin sequence comprises a heavychain variable domain comprising at least or about 100% sequenceidentity to any one of SEQ ID NOs: 138-141. In some instances, the CD3antibody or immunoglobulin sequence comprises a heavy chain variabledomain comprising at least a portion having at least or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, or more than 400 amino acids of any one of SEQ ID NOs:138-141.

In some instances, the CD3 antibody or immunoglobulin sequence comprisesa light chain variable domain comprising at least or about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 142-145. In some instances, the CD3antibody or immunoglobulin sequence comprises a light chain variabledomain comprising at least or about 95% sequence identity to any one ofSEQ ID NOs: 142-145. In some instances, the CD3 antibody orimmunoglobulin sequence comprises a light chain variable domaincomprising at least or about 97% sequence identity to any one of SEQ IDNOs: 142-145. In some instances, the CD3 antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least orabout 99% sequence identity to any one of SEQ ID NOs: 142-145. In someinstances, the CD3 antibody or immunoglobulin sequence comprises a lightchain variable domain comprising at least or about 100% sequenceidentity to any one of SEQ ID NOs: 142-145. In some instances, the CD3antibody or immunoglobulin sequence comprises a light chain variabledomain comprising at least a portion having at least or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, or more than 400 amino acids of any one of SEQ ID NOs:142-145.

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a pluralityof nucleic acids, wherein each nucleic acid encodes for a variant codonsequence compared to a reference nucleic acid sequence. In someinstances, each nucleic acid of a first nucleic acid population containsa variant at a single variant site. In some instances, the first nucleicacid population contains a plurality of variants at a single variantsite such that the first nucleic acid population contains more than onevariant at the same variant site. The first nucleic acid population maycomprise nucleic acids collectively encoding multiple codon variants atthe same variant site. The first nucleic acid population may comprisenucleic acids collectively encoding up to 19 or more codons at the sameposition. The first nucleic acid population may comprise nucleic acidscollectively encoding up to 60 variant triplets at the same position, orthe first nucleic acid population may comprise nucleic acidscollectively encoding up to 61 different triplets of codons at the sameposition. Each variant may encode for a codon that results in adifferent amino acid during translation. Table 1B provides a listing ofeach codon possible (and the representative amino acid) for a variantsite.

TABLE 1B List of codons and amino acids One Three letter letter AminoAcids code code Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGCTGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAGPhenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine HHis CAC CAT Isoleucine I Iso ATA ATC ATT Lysine K Lys AAA AAG Leucine LLeu TTA TTG CTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AACAAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R ArgAGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine TThr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGGTyrosine Y Tyr TAC TAT

A nucleic acid population may comprise varied nucleic acids collectivelyencoding up to 20 codon variations at multiple positions. In such cases,each nucleic acid in the population comprises variation for codons atmore than one position in the same nucleic acid. In some instances, eachnucleic acid in the population comprises variation for codons at 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morecodons in a single nucleic acid. In some instances, each variant longnucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more codons in a single long nucleic acid. In someinstances, the variant nucleic acid population comprises variation forcodons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in asingle nucleic acid. In some instances, the variant nucleic acidpopulation comprises variation for codons in at least about 10, 20, 30,40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleicacid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization,parallelization, and vertical integration of the end-to-end process frompolynucleotide synthesis to gene assembly within nanowells on silicon tocreate a revolutionary synthesis platform. Devices described hereinprovide, with the same footprint as a 96-well plate, a silicon synthesisplatform is capable of increasing throughput by a factor of up to 1,000or more compared to traditional synthesis methods, with production of upto approximately 1,000,000 or more polynucleotides, or 10,000 or moregenes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomicdata has become an important factor for studies that delve into thebiological roles of various genes in both normal biology and diseasepathogenesis. At the core of this research is the central dogma ofmolecular biology and the concept of “residue-by-residue transfer ofsequential information.” Genomic information encoded in the DNA istranscribed into a message that is then translated into the protein thatis the active product within a given biological pathway.

Another exciting area of study is on the discovery, development andmanufacturing of therapeutic molecules focused on a highly-specificcellular target. High diversity DNA sequence libraries are at the coreof development pipelines for targeted therapeutics. Gene mutants areused to express proteins in a design, build, and test proteinengineering cycle that ideally culminates in an optimized gene for highexpression of a protein with high affinity for its therapeutic target.As an example, consider the binding pocket of a receptor. The ability totest all sequence permutations of all residues within the binding pocketsimultaneously will allow for a thorough exploration, increasing chancesof success. Saturation mutagenesis, in which a researcher attempts togenerate all possible mutations at a specific site within the receptor,represents one approach to this development challenge. Though costly andtime and labor-intensive, it enables each variant to be introduced intoeach position. In contrast, combinatorial mutagenesis, where a fewselected positions or short stretch of DNA may be modified extensively,generates an incomplete repertoire of variants with biasedrepresentation.

To accelerate the drug development pipeline, a library with the desiredvariants available at the intended frequency in the right positionavailable for testing—in other words, a precision library, enablesreduced costs as well as turnaround time for screening. Provided hereinare methods for synthesizing nucleic acid synthetic variant librarieswhich provide for precise introduction of each intended variant at thedesired frequency. To the end user, this translates to the ability tonot only thoroughly sample sequence space but also be able to querythese hypotheses in an efficient manner, reducing cost and screeningtime. Genome-wide editing can elucidate important pathways, librarieswhere each variant and sequence permutation can be tested for optimalfunctionality, and thousands of genes can be used to reconstruct entirepathways and genomes to re-engineer biological systems for drugdiscovery.

In a first example, a drug itself can be optimized using methodsdescribed herein. For example, to improve a specified function of anantibody, a variant polynucleotide library encoding for a portion of theantibody is designed and synthesized. A variant nucleic acid library forthe antibody can then be generated by processes described herein (e.g.,PCR mutagenesis followed by insertion into a vector). The antibody isthen expressed in a production cell line and screened for enhancedactivity. Example screens include examining modulation in bindingaffinity to an antigen, stability, or effector function (e.g., ADCC,complement, or apoptosis). Exemplary regions to optimize the antibodyinclude, without limitation, the Fc region, Fab region, variable regionof the Fab region, constant region of the Fab region, variable domain ofthe heavy chain or light chain (V_(H) or V_(L)), and specificcomplementarity-determining regions (CDRs) of V_(H) or V_(L).

Nucleic acid libraries synthesized by methods described herein may beexpressed in various cells associated with a disease state. Cellsassociated with a disease state include cell lines, tissue samples,primary cells from a subject, cultured cells expanded from a subject, orcells in a model system. Exemplary model systems include, withoutlimitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction ortreatment of a disease state, a variant nucleic acid library describedherein is expressed in a cell associated with a disease state, or one inwhich a cell a disease state can be induced. In some instances, an agentis used to induce a disease state in cells. Exemplary tools for diseasestate induction include, without limitation, a Cre/Lox recombinationsystem, LPS inflammation induction, and streptozotocin to inducehypoglycemia. The cells associated with a disease state may be cellsfrom a model system or cultured cells, as well as cells from a subjecthaving a particular disease condition. Exemplary disease conditionsinclude a bacterial, fungal, viral, autoimmune, or proliferativedisorder (e.g., cancer). In some instances, the variant nucleic acidlibrary is expressed in the model system, cell line, or primary cellsderived from a subject, and screened for changes in at least onecellular activity. Exemplary cellular activities include, withoutlimitation, proliferation, cycle progression, cell death, adhesion,migration, reproduction, cell signaling, energy production, oxygenutilization, metabolic activity, and aging, response to free radicaldamage, or any combination thereof.

Substrates

Devices used as a surface for polynucleotide synthesis may be in theform of substrates which include, without limitation, homogenous arraysurfaces, patterned array surfaces, channels, beads, gels, and the like.Provided herein are substrates comprising a plurality of clusters,wherein each cluster comprises a plurality of loci that support theattachment and synthesis of polynucleotides. In some instances,substrates comprise a homogenous array surface. For example, thehomogenous array surface is a homogenous plate. The term “locus” as usedherein refers to a discrete region on a structure which provides supportfor polynucleotides encoding for a single predetermined sequence toextend from the surface. In some instances, a locus is on a twodimensional surface, e.g., a substantially planar surface. In someinstances, a locus is on a three-dimensional surface, e.g., a well,microwell, channel, or post. In some instances, a surface of a locuscomprises a material that is actively functionalized to attach to atleast one nucleotide for polynucleotide synthesis, or preferably, apopulation of identical nucleotides for synthesis of a population ofpolynucleotides. In some instances, polynucleotide refers to apopulation of polynucleotides encoding for the same nucleic acidsequence. In some cases, a surface of a substrate is inclusive of one ora plurality of surfaces of a substrate. The average error rates forpolynucleotides synthesized within a library described here using thesystems and methods provided are often less than 1 in 1000, less thanabout 1 in 2000, less than about 1 in 3000 or less often without errorcorrection.

Provided herein are surfaces that support the parallel synthesis of aplurality of polynucleotides having different predetermined sequences ataddressable locations on a common support. In some instances, asubstrate provides support for the synthesis of more than 50, 100, 200,400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000;20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000;1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000;4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides.In some cases, the surfaces provide support for the synthesis of morethan 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000;5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000;500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000;1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000;3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or morepolynucleotides encoding for distinct sequences. In some instances, atleast a portion of the polynucleotides have an identical sequence or areconfigured to be synthesized with an identical sequence. In someinstances, the substrate provides a surface environment for the growthof polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinctloci of a substrate, wherein each locus supports the synthesis of apopulation of polynucleotides. In some cases, each locus supports thesynthesis of a population of polynucleotides having a different sequencethan a population of polynucleotides grown on another locus. In someinstances, each polynucleotide sequence is synthesized with 1, 2, 3, 4,5, 6, 7, 8, 9 or more redundancy across different loci within the samecluster of loci on a surface for polynucleotide synthesis. In someinstances, the loci of a substrate are located within a plurality ofclusters. In some instances, a substrate comprises at least 10, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000,12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.In some instances, a substrate comprises more than 2,000; 5,000; 10,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000;1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or10,000,000 or more distinct loci. In some instances, a substratecomprises about 10,000 distinct loci. The amount of loci within a singlecluster is varied in different instances. In some cases, each clusterincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances,each cluster includes about 50-500 loci. In some instances, each clusterincludes about 100-200 loci. In some instances, each cluster includesabout 100-150 loci. In some instances, each cluster includes about 109,121, 130 or 137 loci. In some instances, each cluster includes about 19,20, 61, 64 or more loci. Alternatively or in combination, polynucleotidesynthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized ona substrate is dependent on the number of distinct loci available in thesubstrate. In some instances, the density of loci within a cluster orsurface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100,130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In somecases, a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500,10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distancebetween the centers of two adjacent loci within a cluster or surface isfrom about 10-500, from about 10-200, or from about 10-100 um. In someinstances, the distance between two centers of adjacent loci is greaterthan about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In someinstances, the distance between the centers of two adjacent loci is lessthan about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, eachlocus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is atleast or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 clusterper 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50clusters per 1 mm² or more. In some instances, a substrate comprisesfrom about 1 cluster per 10 mm² to about 10 clusters per 1 mm². In someinstances, the distance between the centers of two adjacent clusters isat least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10,0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, eachcluster has a cross section of about 0.5 to about 2, about 0.5 to about1, or about 1 to about 2 mm. In some cases, each cluster has a crosssection of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interiorcross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 wellplate, for example between about 100 and about 200 mm by between about50 and about 150 mm. In some instances, a substrate has a diameter lessthan or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or50 mm. In some instances, the diameter of a substrate is between about25-1000, 25-800, 25-600, 25-500, 25-400, 25-300, or 25-200 mm. In someinstances, a substrate has a planar surface area of at least about 100;200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000;40,000; 50,000 mm² or more. In some instances, the thickness of asubstrate is between about 50-2000, 50-1000, 100-1000, 200-1000, or250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated fromany variety of materials suitable for the methods, compositions, andsystems described herein. In certain instances, substrate materials arefabricated to exhibit a low level of nucleotide binding. In someinstances, substrate materials are modified to generate distinctsurfaces that exhibit a high level of nucleotide binding. In someinstances, substrate materials are transparent to visible and/or UVlight. In some instances, substrate materials are sufficientlyconductive, e.g., are able to form uniform electric fields across all ora portion of a substrate. In some instances, conductive materials areconnected to an electric ground. In some instances, the substrate isheat conductive or insulated. In some instances, the materials arechemical resistant and heat resistant to support chemical or biochemicalreactions, for example polynucleotide synthesis reaction processes. Insome instances, a substrate comprises flexible materials. For flexiblematerials, materials can include, without limitation: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like. Insome instances, a substrate comprises rigid materials. For rigidmaterials, materials can include, without limitation: glass; fusesilica; silicon, plastics (for example polytetraflouroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). Thesubstrate, solid support or reactors can be fabricated from a materialselected from the group consisting of silicon, polystyrene, agarose,dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane(PDMS), and glass. The substrates/solid supports or the microstructures,reactors therein may be manufactured with a combination of materialslisted herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates have a surfacearchitecture suitable for the methods, compositions, and systemsdescribed herein. In some instances, a substrate comprises raised and/orlowered features. One benefit of having such features is an increase insurface area to support polynucleotide synthesis. In some instances, asubstrate having raised and/or lowered features is referred to as athree-dimensional substrate. In some cases, a three-dimensionalsubstrate comprises one or more channels. In some cases, one or moreloci comprise a channel. In some cases, the channels are accessible toreagent deposition via a deposition device such as a material depositiondevice. In some cases, reagents and/or fluids collect in a larger wellin fluid communication one or more channels. For example, a substratecomprises a plurality of channels corresponding to a plurality of lociwith a cluster, and the plurality of channels are in fluid communicationwith one well of the cluster. In some methods, a library ofpolynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates are configured forpolynucleotide synthesis. In some instances, the structure is configuredto allow for controlled flow and mass transfer paths for polynucleotidesynthesis on a surface. In some instances, the configuration of asubstrate allows for the controlled and even distribution of masstransfer paths, chemical exposure times, and/or wash efficacy duringpolynucleotide synthesis. In some instances, the configuration of asubstrate allows for increased sweep efficiency, for example byproviding sufficient volume for a growing polynucleotide such that theexcluded volume by the growing polynucleotide does not take up more than50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, 1%, or less of the initially available volume that is available orsuitable for growing the polynucleotide. In some instances, athree-dimensional structure allows for managed flow of fluid to allowfor the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates comprise structuressuitable for the methods, compositions, and systems described herein. Insome instances, segregation is achieved by physical structure. In someinstances, segregation is achieved by differential functionalization ofthe surface generating active and passive regions for polynucleotidesynthesis. In some instances, differential functionalization is achievedby alternating the hydrophobicity across the substrate surface, therebycreating water contact angle effects that cause beading or wetting ofthe deposited reagents. Employing larger structures can decreasesplashing and cross-contamination of distinct polynucleotide synthesislocations with reagents of the neighboring spots. In some cases, adevice, such as a material deposition device, is used to depositreagents to distinct polynucleotide synthesis locations. Substrateshaving three-dimensional features are configured in a manner that allowsfor the synthesis of a large number of polynucleotides (e.g., more thanabout 10,000) with a low error rate (e.g., less than about 1:500,1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases,a substrate comprises features with a density of about or greater thanabout 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height,and/or volume as another well of the substrate. A channel of a substratemay have the same or different width, height, and/or volume as anotherchannel of the substrate. In some instances, the diameter of a clusteror the diameter of a well comprising a cluster, or both, is betweenabout 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1,0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or0.5-2 mm. In some instances, the diameter of a cluster or well or bothis less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06,or 0.05 mm. In some instances, the diameter of a cluster or well or bothis between about 1.0 and 1.3 mm. In some instances, the diameter of acluster or well, or both is about 1.150 mm. In some instances, thediameter of a cluster or well, or both is about 0.08 mm. The diameter ofa cluster refers to clusters within a two-dimensional orthree-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000,100-1000, 200-1000, 300-1000, 400-1000, or 500-1000 um. In some cases,the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channelscorresponding to a plurality of loci within a cluster, wherein theheight or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50,or 10-50 um. In some cases, the height of a channel is less than 100,80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in asubstantially planar substrate) or both channel and locus (e.g., in athree-dimensional substrate wherein a locus corresponds to a channel) isfrom about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, forexample, about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the diameter of a channel, locus, or both channel and locusis less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the distance between the center of two adjacent channels,loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200,5-100, 5-50, or 5-30, for example, about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface,wherein the surface comprises various surface modifications. In someinstances, the surface modifications are employed for the chemicaland/or physical alteration of a surface by an additive or subtractiveprocess to change one or more chemical and/or physical properties of asubstrate surface or a selected site or region of a substrate surface.For example, surface modifications include, without limitation, (1)changing the wetting properties of a surface, (2) functionalizing asurface, i.e., providing, modifying or substituting surface functionalgroups, (3) defunctionalizing a surface, i.e., removing surfacefunctional groups, (4) otherwise altering the chemical composition of asurface, e.g., through etching, (5) increasing or decreasing surfaceroughness, (6) providing a coating on a surface, e.g., a coating thatexhibits wetting properties that are different from the wettingproperties of the surface, and/or (7) depositing particulates on asurface.

In some cases, the addition of a chemical layer on top of a surface(referred to as adhesion promoter) facilitates structured patterning ofloci on a surface of a substrate. Exemplary surfaces for application ofadhesion promotion include, without limitation, glass, silicon, silicondioxide and silicon nitride. In some cases, the adhesion promoter is achemical with a high surface energy. In some instances, a secondchemical layer is deposited on a surface of a substrate. In some cases,the second chemical layer has a low surface energy. In some cases,surface energy of a chemical layer coated on a surface supportslocalization of droplets on the surface. Depending on the patterningarrangement selected, the proximity of loci and/or area of fluid contactat the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto whichnucleic acids or other moieties are deposited, e.g., for polynucleotidesynthesis, are smooth or substantially planar (e.g., two-dimensional) orhave irregularities, such as raised or lowered features (e.g.,three-dimensional features). In some instances, a substrate surface ismodified with one or more different layers of compounds. Suchmodification layers of interest include, without limitation, inorganicand organic layers such as metals, metal oxides, polymers, small organicmolecules and the like.

In some instances, resolved loci of a substrate are functionalized withone or more moieties that increase and/or decrease surface energy. Insome cases, a moiety is chemically inert. In some cases, a moiety isconfigured to support a desired chemical reaction, for example, one ormore processes in a polynucleotide synthesis reaction. The surfaceenergy, or hydrophobicity, of a surface is a factor for determining theaffinity of a nucleotide to attach onto the surface. In some instances,a method for substrate functionalization comprises: (a) providing asubstrate having a surface that comprises silicon dioxide; and (b)silanizing the surface using, a suitable silanizing agent describedherein or otherwise known in the art, for example, an organofunctionalalkoxysilane molecule. Methods and functionalizing agents are describedin U.S. Pat. No. 5,474,796, which is herein incorporated by reference inits entirety.

In some instances, a substrate surface is functionalized by contact witha derivatizing composition that contains a mixture of silanes, underreaction conditions effective to couple the silanes to the substratesurface, typically via reactive hydrophilic moieties present on thesubstrate surface. Silanization generally covers a surface throughself-assembly with organofunctional alkoxysilane molecules. A variety ofsiloxane functionalizing reagents can further be used as currently knownin the art, e.g., for lowering or increasing surface energy. Theorganofunctional alkoxysilanes are classified according to their organicfunctions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis mayinclude processes involving phosphoramidite chemistry. In someinstances, polynucleotide synthesis comprises coupling a base withphosphoramidite. Polynucleotide synthesis may comprise coupling a baseby deposition of phosphoramidite under coupling conditions, wherein thesame base is optionally deposited with phosphoramidite more than once,i.e., double coupling. Polynucleotide synthesis may comprise capping ofunreacted sites. In some instances, capping is optional. Polynucleotidesynthesis may also comprise oxidation or an oxidation step or oxidationsteps. Polynucleotide synthesis may comprise deblocking, detritylation,and sulfurization. In some instances, polynucleotide synthesis compriseseither oxidation or sulfurization. In some instances, between one oreach step during a polynucleotide synthesis reaction, the device iswashed, for example, using tetrazole or acetonitrile. Time frames forany one step in a phosphoramidite synthesis method may be less thanabout 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise asubsequent addition of a phosphoramidite building block (e.g.,nucleoside phosphoramidite) to a growing polynucleotide chain for theformation of a phosphite triester linkage. Phosphoramiditepolynucleotide synthesis proceeds in the 3′ to 5′ direction.Phosphoramidite polynucleotide synthesis allows for the controlledaddition of one nucleotide to a growing nucleic acid chain per synthesiscycle. In some instances, each synthesis cycle comprises a couplingstep. Phosphoramidite coupling involves the formation of a phosphitetriester linkage between an activated nucleoside phosphoramidite and anucleoside bound to the substrate, for example, via a linker. In someinstances, the nucleoside phosphoramidite is provided to the deviceactivated. In some instances, the nucleoside phosphoramidite is providedto the device with an activator. In some instances, nucleosidephosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100-fold excess or more over the substrate-boundnucleosides. In some instances, the addition of nucleosidephosphoramidite is performed in an anhydrous environment, for example,in anhydrous acetonitrile. Following addition of a nucleosidephosphoramidite, the device is optionally washed. In some instances, thecoupling step is repeated one or more additional times, optionally witha wash step between nucleoside phosphoramidite additions to thesubstrate. In some instances, a polynucleotide synthesis method usedherein comprises 1, 2, 3 or more sequential coupling steps. Prior tocoupling, in many cases, the nucleoside bound to the device isde-protected by removal of a protecting group, where the protectinggroup functions to prevent polymerization. A common protecting group is4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingpolynucleotide is treated with a capping agent. A capping step is usefulto block unreacted substrate-bound 5′-OH groups after coupling fromfurther chain elongation, preventing the formation of polynucleotideswith internal base deletions. Further, phosphoramidites activated with1H-tetrazole may react, to a small extent, with the O6 position ofguanosine. Without being bound by theory, upon oxidation with I₂/water,this side product, possibly via O6-N7 migration, may undergodepurination. The apurinic sites may end up being cleaved in the courseof the final deprotection of the polynucleotide thus reducing the yieldof the full-length product. The O6 modifications may be removed bytreatment with the capping reagent prior to oxidation with I2/water. Insome instances, inclusion of a capping step during polynucleotidesynthesis decreases the error rate as compared to synthesis withoutcapping. As an example, the capping step comprises treating thesubstrate-bound polynucleotide with a mixture of acetic anhydride and1-methylimidazole. Following a capping step, the device is optionallywashed.

In some instances, following addition of a nucleoside phosphoramidite,and optionally after capping and one or more wash steps, the devicebound growing nucleic acid is oxidized. The oxidation step comprises thephosphite triester is oxidized into a tetracoordinated phosphatetriester, a protected precursor of the naturally occurring phosphatediester internucleoside linkage. In some instances, oxidation of thegrowing polynucleotide is achieved by treatment with iodine and water,optionally in the presence of a weak base (e.g., pyridine, lutidine,collidine). Oxidation may be carried out under anhydrous conditionsusing, e.g. tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for device drying, as residual water from oxidation that maypersist can inhibit subsequent coupling. Following oxidation, the deviceand growing polynucleotide is optionally washed. In some instances, thestep of oxidation is substituted with a sulfurization step to obtainpolynucleotide phosphorothioates, wherein any capping steps can beperformed after the sulfurization. Many reagents are capable of theefficient sulfur transfer, including but not limited to3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occurthrough coupling, the protected 5′ end of the device bound growingpolynucleotide is removed so that the primary hydroxyl group is reactivewith a next nucleoside phosphoramidite. In some instances, theprotecting group is DMT and deblocking occurs with trichloroacetic acidin dichloromethane. Conducting detritylation for an extended time orwith stronger than recommended solutions of acids may lead to increaseddepurination of solid support-bound polynucleotide and thus reduces theyield of the desired full-length product. Methods and compositions ofthe disclosure described herein provide for controlled deblockingconditions limiting undesired depurination reactions. In some instances,the device bound polynucleotide is washed after deblocking. In someinstances, efficient washing after deblocking contributes to synthesizedpolynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve aniterating sequence of the following steps: application of a protectedmonomer to an actively functionalized surface (e.g., locus) to link witheither the activated surface, a linker or with a previously deprotectedmonomer; deprotection of the applied monomer so that it is reactive witha subsequently applied protected monomer; and application of anotherprotected monomer for linking. One or more intermediate steps includeoxidation or sulfurization. In some instances, one or more wash stepsprecede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise aseries of chemical steps. In some instances, one or more steps of asynthesis method involve reagent cycling, where one or more steps of themethod comprise application to the device of a reagent useful for thestep. For example, reagents are cycled by a series of liquid depositionand vacuum drying steps. For substrates comprising three-dimensionalfeatures such as wells, microwells, channels and the like, reagents areoptionally passed through one or more regions of the device via thewells and/or channels.

Methods and systems described herein relate to polynucleotide synthesisdevices for the synthesis of polynucleotides. The synthesis may be inparallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or morepolynucleotides can be synthesized in parallel. The total numberpolynucleotides that may be synthesized in parallel may be from2-100000, 3-50000, 4-10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700,11-650, 12-600, 13-550, 14-500, 15-450, 16-400, 17-350, 18-300, 19-250,20-200, 21-150,22-100, 23-50, 24-45, 25-40, 30-35. Those of skill in theart appreciate that the total number of polynucleotides synthesized inparallel may fall within any range bound by any of these values, forexample 25-100. The total number of polynucleotides synthesized inparallel may fall within any range defined by any of the values servingas endpoints of the range. Total molar mass of polynucleotidessynthesized within the device or the molar mass of each of thepolynucleotides may be at least or at least about 10, 20, 30, 40, 50,100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The lengthof each of the polynucleotides or average length of the polynucleotideswithin the device may be at least or about at least 10, 15, 20, 25, 30,35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. Thelength of each of the polynucleotides or average length of thepolynucleotides within the device may be at most or about at most 500,400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10 nucleotides, or less. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100,15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciatethat the length of each of the polynucleotides or average length of thepolynucleotides within the device may fall within any range bound by anyof these values, for example 100-300. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall within any range defined by any of the values serving asendpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allowfor synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175,200 nucleotides per hour, or more are synthesized. Nucleotides includeadenine, guanine, thymine, cytosine, uridine building blocks, oranalogs/modified versions thereof. In some instances, libraries ofpolynucleotides are synthesized in parallel on substrate. For example, adevice comprising about or at least about 100; 1,000; 10,000; 30,000;75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or5,000,000 resolved loci is able to support the synthesis of at least thesame number of distinct polynucleotides, wherein polynucleotide encodinga distinct sequence is synthesized on a resolved locus. In someinstances, a library of polynucleotides is synthesized on a device withlow error rates described herein in less than about three months, twomonths, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acidsassembled from a polynucleotide library synthesized with low error rateusing the substrates and methods described herein are prepared in lessthan about three months, two months, one month, three weeks, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of alibrary of nucleic acids comprising variant nucleic acids differing at aplurality of codon sites. In some instances, a nucleic acid may have 1site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may beadjacent. In some instances, the one or more sites of variant codonsites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variantcodon sites, wherein all the variant codon sites are adjacent to oneanother, forming a stretch of variant codon sites. In some instances, anucleic acid may comprise multiple sites of variant codon sites, whereinnone the variant codon sites are adjacent to one another. In someinstances, a nucleic acid may comprise multiple sites of variant codonsites, wherein some the variant codon sites are adjacent to one another,forming a stretch of variant codon sites, and some of the variant codonsites are not adjacent to one another.

Referring to the Figures, FIG. 1 illustrates an exemplary processworkflow for synthesis of nucleic acids (e.g., genes) from shorternucleic acids. The workflow is divided generally into phases: (1) denovo synthesis of a single stranded nucleic acid library, (2) joiningnucleic acids to form larger fragments, (3) error correction, (4)quality control, and (5) shipment. Prior to de novo synthesis, anintended nucleic acid sequence or group of nucleic acid sequences ispreselected. For example, a group of genes is preselected forgeneration.

Once large nucleic acids for generation are selected, a predeterminedlibrary of nucleic acids is designed for de novo synthesis. Varioussuitable methods are known for generating high density polynucleotidearrays. In the workflow example, a device surface layer is provided. Inthe example, chemistry of the surface is altered in order to improve thepolynucleotide synthesis process. Areas of low surface energy aregenerated to repel liquid while areas of high surface energy aregenerated to attract liquids. The surface itself may be in the form of aplanar surface or contain variations in shape, such as protrusions ormicrowells which increase surface area. In the workflow example, highsurface energy molecules selected serve a dual function of supportingDNA chemistry, as disclosed in International Patent ApplicationPublication WO/2015/021080, which is herein incorporated by reference inits entirety.

In situ preparation of polynucleotide arrays is generated on a solidsupport and utilizes single nucleotide extension process to extendmultiple oligomers in parallel. A deposition device, such as a materialdeposition device, is designed to release reagents in a step wisefashion such that multiple polynucleotides extend, in parallel, oneresidue at a time to generate oligomers with a predetermined nucleicacid sequence 102. In some instances, polynucleotides are cleaved fromthe surface at this stage. Cleavage includes gas cleavage, e. g., withammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber.In this exemplary workflow, the reaction chamber (also referred to as“nanoreactor”) is a silicon coated well, containing PCR reagents andlowered onto the polynucleotide library 103. Prior to or after thesealing 104 of the polynucleotides, a reagent is added to release thepolynucleotides from the substrate. In the exemplary workflow, thepolynucleotides are released subsequent to sealing of the nanoreactor105. Once released, fragments of single stranded polynucleotideshybridize in order to span an entire long range sequence of DNA. Partialhybridization 105 is possible because each synthesized polynucleotide isdesigned to have a small portion overlapping with at least one otherpolynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerasecycles, the polynucleotides anneal to complementary fragments and gapsare filled in by a polymerase. Each cycle increases the length ofvarious fragments randomly depending on which polynucleotides find eachother. Complementarity amongst the fragments allows for forming acomplete large span of double stranded DNA 106.

After PCA is complete, the nanoreactor is separated from the device 107and positioned for interaction with a device having primers for PCR 108.After sealing, the nanoreactor is subject to PCR 109 and the largernucleic acids are amplified. After PCR 110, the nanochamber is opened111, error correction reagents are added 112, the chamber is sealed 113and an error correction reaction occurs to remove mismatched base pairsand/or strands with poor complementarity from the double stranded PCRamplification products 114. The nanoreactor is opened and separated 115.Error corrected product is next subject to additional processing steps,such as PCR and molecular bar coding, and then packaged 122 for shipment123.

In some instances, quality control measures are taken. After errorcorrection, quality control steps include for example interaction with awafer having sequencing primers for amplification of the error correctedproduct 116, sealing the wafer to a chamber containing error correctedamplification product 117, and performing an additional round ofamplification 118. The nanoreactor is opened 119 and the products arepooled 120 and sequenced 121. After an acceptable quality controldetermination is made, the packaged product 122 is approved for shipment123.

In some instances, a nucleic acid generated by a workflow such as thatin FIG. 1 is subject to mutagenesis using overlapping primers disclosedherein. In some instances, a library of primers are generated by in situpreparation on a solid support and utilize single nucleotide extensionprocess to extend multiple oligomers in parallel. A deposition device,such as a material deposition device, is designed to release reagents ina step wise fashion such that multiple polynucleotides extend, inparallel, one residue at a time to generate oligomers with apredetermined nucleic acid sequence 102.

Computer systems

Any of the systems described herein, may be operably linked to acomputer and may be automated through a computer either locally orremotely. In various instances, the methods and systems of thedisclosure may further comprise software programs on computer systemsand use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of thedisclosure. The computer systems may be programmed to interface betweenthe user specified base sequence and the position of a materialdeposition device to deliver the correct reagents to specified regionsof the substrate.

The computer system 200 illustrated in FIG. 2 may be understood as alogical apparatus that can read instructions from media 211 and/or anetwork port 205, which can optionally be connected to server 209 havingfixed media 212. The system, such as shown in FIG. 2 can include a CPU201, disk drives 203, optional input devices such as keyboard 215 and/ormouse 216 and optional monitor 207. Data communication can be achievedthrough the indicated communication medium to a server at a local or aremote location. The communication medium can include any means oftransmitting and/or receiving data. For example, the communicationmedium can be a network connection, a wireless connection or an internetconnection. Such a connection can provide for communication over theWorld Wide Web. It is envisioned that data relating to the presentdisclosure can be transmitted over such networks or connections forreception and/or review by a party 222 as illustrated in FIG. 2.

As illustrated in FIG. 3, a high speed cache 304 can be connected to, orincorporated in, the processor 302 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 302. The processor 302 is connected to a north bridge 306 by aprocessor bus 308. The north bridge 306 is connected to random accessmemory (RAM) 310 by a memory bus 312 and manages access to the RAM 310by the processor 302. The north bridge 306 is also connected to a southbridge 314 by a chipset bus 316. The south bridge 314 is, in turn,connected to a peripheral bus 318. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 318. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip. In someinstances, system 300 can include an accelerator card 322 attached tothe peripheral bus 318. The accelerator can include field programmablegate arrays (FPGAs) or other hardware for accelerating certainprocessing. For example, an accelerator can be used for adaptive datarestructuring or to evaluate algebraic expressions used in extended setprocessing.

Software and data are stored in external storage 324 and can be loadedinto RAM 310 and/or cache 304 for use by the processor. The system 300includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MACOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample instances of the present disclosure. In this example, system 300also includes network interface cards (NICs) 320 and 321 connected tothe peripheral bus for providing network interfaces to external storage,such as Network Attached Storage (NAS) and other computer systems thatcan be used for distributed parallel processing.

FIG. 4 is a diagram showing a network 400 with a plurality of computersystems 402 a, and 402 b, a plurality of cell phones and personal dataassistants 402 c, and Network Attached Storage (NAS) 404 a, and 404 b.In example instances, systems 402 a, 402 b, and 402 c can manage datastorage and optimize data access for data stored in Network AttachedStorage (NAS) 404 a and 404 b. A mathematical model can be used for thedata and be evaluated using distributed parallel processing acrosscomputer systems 402 a, and 402 b, and cell phone and personal dataassistant systems 402 c. Computer systems 402 a, and 402 b, and cellphone and personal data assistant systems 402 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 404 a and 404 b. FIG. 4 illustrates anexample only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various instances of thepresent disclosure. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface. In some example instances, processors can maintainseparate memory spaces and transmit data through network interfaces,back plane or other connectors for parallel processing by otherprocessors. In other instances, some or all of the processors can use ashared virtual address memory space.

FIG. 5 is a block diagram of a multiprocessor computer system 500 usinga shared virtual address memory space in accordance with an exampleinstance. The system includes a plurality of processors 502 a-f that canaccess a shared memory subsystem 504. The system incorporates aplurality of programmable hardware memory algorithm processors (MAPs)506 a-f in the memory subsystem 504. Each MAP 506 a-f can comprise amemory 508 a-f and one or more field programmable gate arrays (FPGAs)510 a-f. The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms can be provided to the FPGAs 510a-f for processing in close coordination with a respective processor.For example, the MAPs can be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample instances. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPcan use Direct Memory Access (DMA) to access an associated memory 508a-f, allowing it to execute tasks independently of, and asynchronouslyfrom the respective microprocessor 502 a-f. In this configuration, a MAPcan feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleinstances, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some instances, allor part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example instances, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other instances, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 3, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 322 illustrated in FIG. 3.

The following examples are set forth to illustrate more clearly theprinciple and practice of embodiments disclosed herein to those skilledin the art and are not to be construed as limiting the scope of anyclaimed embodiments. Unless otherwise stated, all parts and percentagesare on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of thedisclosure. Changes therein and other uses which are encompassed withinthe spirit of the disclosure as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of alibrary of polynucleotides. The device surface was first wet cleanedusing a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20minutes. The device was rinsed in several beakers with DI water, heldunder a DI water gooseneck faucet for 5 min, and dried with N₂. Thedevice was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min,rinsed with DI water using a handgun, soaked in three successive beakerswith DI water for 1 min each, and then rinsed again with DI water usingthe handgun. The device was then plasma cleaned by exposing the devicesurface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solutioncomprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using aYES-1224P vapor deposition oven system with the following parameters:0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface wasresist coated using a Brewer Science 200X spin coater. SPR™ 3612photoresist was spin coated on the device at 2500 rpm for 40 sec. Thedevice was pre-baked for 30 min at 90° C. on a Brewer hot plate. Thedevice was subjected to photolithography using a Karl Suss MA6 maskaligner instrument. The device was exposed for 2.2 sec and developed for1 min in MSF 26A. Remaining developer was rinsed with the handgun andthe device soaked in water for 5 min. The device was baked for 30 min at100° C. in the oven, followed by visual inspection for lithographydefects using a Nikon L200. A descum process was used to remove residualresist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 wattsfor 1 min.

The device surface was passively functionalized with a 100 μL solutionof perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. Thedevice was placed in a chamber, pumped for 10 min, and then the valvewas closed to the pump and left to stand for 10 min. The chamber wasvented to air. The device was resist stripped by performing two soaksfor 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power(9 on Crest system). The device was then soaked for 5 min in 500 mLisopropanol at room temperature with ultrasonication at maximum power.The device was dipped in 300 mL of 200 proof ethanol and blown dry withN₂. The functionalized surface was activated to serve as a support forpolynucleotide synthesis.

Example 2: Synthesis of a 50-mer Sequence on an OligonucleotideSynthesis Device

A two dimensional oligonucleotide synthesis device was assembled into aflowcell, which was connected to a flowcell (Applied Biosystems (ABI394DNA Synthesizer”). The two-dimensional oligonucleotide synthesis devicewas uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used tosynthesize an exemplary polynucleotide of 50 bp (“50-merpolynucleotide”) using polynucleotide synthesis methods describedherein.

The sequence of the 50-mer was as described in SEQ ID NO. 104.5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3′ (SEQID NO. 104), where # denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 2and an ABI synthesizer.

TABLE 2 Synthesis protocol General DNA Synthesis Table 2 Process NameProcess Step Time (sec) WASH (Acetonitrile Wash Acetonitrile SystemFlush 4 Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE ADDITION Activator Manifold Flush 2(Phosphoramidite + Activator to Flowcell 6 Activator Flow) Activator + 6Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Incubate for 25 sec 25 WASH (AcetonitrileWash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION ActivatorManifold Flush 2 (Phosphoramidite + Activator to Flowcell 5 ActivatorFlow) Activator + 18 Phosphoramidite to Flowcell Incubate for 25 sec 25WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrileto Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 CAPPING(CapA + B, 1:1, CapA + B to Flowcell 15 Flow) WASH (Acetonitrile WashAcetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15Acetonitrile System Flush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 N2 System Flush4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 SystemFlush 4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock toFlowcell 36 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4Flow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile toFlowcell 18 N2 System Flush 4.13 Acetonitrile System Flush 4.13Acetonitrile to Flowcell 15

The phosphoramidite/activator combination was delivered similar to thedelivery of bulk reagents through the flowcell. No drying steps wereperformed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enablefaster flow. Without flow restrictor, flow rates for amidites (0.1M inACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx fromGlenResearch) in ACN), and Ox (0.02M I2 in 20% pyridine, 10% water, and70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and cappingreagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride inTHF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flowrestrictor). The time to completely push out Oxidizer was observed, thetiming for chemical flow times was adjusted accordingly and an extra ACNwash was introduced between different chemicals. After polynucleotidesynthesis, the chip was deprotected in gaseous ammonia overnight at 75psi. Five drops of water were applied to the surface to recoverpolynucleotides. The recovered polynucleotides were then analyzed on aBioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-mer Sequence on an OligonucleotideSynthesis Device

The same process as described in Example 2 for the synthesis of the50-mer sequence was used for the synthesis of a 100-mer polynucleotide(“100-mer polynucleotide”; 5′CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where # denotesThymidine-succinyl hexamide CED phosphoramidite (CLP-2244 fromChemGenes); SEQ ID NO. 105) on two different silicon chips, the firstone uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second onefunctionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane andn-decyltriethoxysilane, and the polynucleotides extracted from thesurface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using aforward (5′ATGCGGGGTTCTCATCATC3; SEQ ID NO. 106) and a reverse(5′CGGGATCCTTATCGTCATCG3′; SEQ ID NO. 107) primer in a 50 uL PCR mix (25uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverseprimer, 1 uL polynucleotide extracted from the surface, and water up to50 uL) using the following thermalcycling program:

98° C., 30 sec

98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles

72° C., 2 min

The PCR products were also run on a BioAnalyzer, demonstrating sharppeaks at the 100-mer position. Next, the PCR amplified samples werecloned, and Sanger sequenced. Table 3 summarizes the results from theSanger sequencing for samples taken from spots 1-5 from chip 1 and forsamples taken from spots 6-10 from chip 2.

TABLE 3 Sequencing results Spot Error rate Cycle efficiency 1 1/763 bp99.87% 2 1/824 bp 99.88% 3 1/780 bp 99.87% 4 1/429 bp 99.77% 5 1/1525 bp99.93% 6 1/1615 bp 99.94% 7 1/531 bp 99.81% 8 1/1769 bp 99.94% 9 1/854bp 99.88% 10 1/1451 bp 99.93%

Thus, the high quality and uniformity of the synthesized polynucleotideswere repeated on two chips with different surface chemistries. Overall,89% of the 100-mers that were sequenced were perfect sequences with noerrors, corresponding to 233 out of 262.

Table 4 summarizes error characteristics for the sequences obtained fromthe polynucleotide samples from spots 1-10.

TABLE 4 Error characteristics Sample ID/Spot OSA_ OSA_ OSA_ OSA_ OSA_OSA_ OSA_ OSA_ OSA_ OSA_ no. 0046/1 0047/2 0048/3 0049/4 0050/5 0051/60052/7 0053/8 0054/9 0055/10 Total 32 32 32 32 32 32 32 32 32 32Sequences Sequencing 25 of 28 27 of 27 26 of 30 21 of 23 25 of 26 29 of30 27 of 31 29 of 31 28 of 29 25 of 28 Quality Oligo 23 of 25 25 of 2722 of 26 18 of 21 24 of 25 25 of 29 22 of 27 28 of 29 26 of 28 20 of 25Quality ROI 2500 2698 2561 2122 2499 2666 2625 2899 2798 2348 MatchCount ROI 2 2 1 3 1 0 2 1 2 1 Mutation ROI Multi 0 0 0 0 0 0 0 0 0 0Base Deletion ROI Small 1 0 0 0 0 0 0 0 0 0 Insertion ROI 0 0 0 0 0 0 00 0 0 Single Base Deletion Large 0 0 1 0 0 1 1 0 0 0 Deletion CountMutation: 2 2 1 2 1 0 2 1 2 1 G > A Mutation: 0 0 0 1 0 0 0 0 0 0 T > CROI Error 3 2 2 3 1 1 3 1 2 1 Count ROI Error Err: ~1 Err: ~1 Err: ~1Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 Rate in 834 in1350 in 1282 in 708 in 2500 in 2667 in 876 in 2900 in 1400 in 2349 ROIMP Err: MP Err: MP Err: MP Err: MP Err: MP Err: MP Err: MP Err: MP Err:MP Err: Minus ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1in Primer 763 824 780 429 1525 1615 531 1769 854 1451 Error Rate

Example 4: VHH Libraries

Synthetic VHH libraries were developed. For the ‘VHH Ratio’ library withtailored CDR diversity, 2391 VHH sequences (iCAN database) were alignedusing Clustal Omega to determine the consensus at each position and theframework was derived from the consensus at each position. The CDRs ofall of the 2391 sequences were analyzed for position-specific variation,and this diversity was introduced in the library design. For the ‘VHHShuffle’ library with shuffled CDR diversity, the iCAN database wasscanned for unique CDRs in the nanobody sequences. 1239 unique CDR1's,1600 unique CDR2's, and 1608 unique CDR3's were identified and theframework was derived from the consensus at each framework positionamongst the 2391 sequences in the iCAN database. Each of the uniqueCDR's was individually synthesized and shuffled in the consensusframework to generate a library with theoretical diversity of 3.2×10 9.The library was then cloned in the phagemid vector using restrictionenzyme digest. For the ‘VHH hShuffle’ library (a synthetic “human” VHHlibrary with shuffled CDR diversity), the iCAN database was scanned forunique CDRs in the nanobody sequences. 1239 unique CDR1's, 1600 uniqueCDR2's, and 1608 unique CDR3's were identified and framework 1, 3, and 4was derived from the human germline DP-47 framework. Framework 2 wasderived from the consensus at each framework position amongst the 2391sequences in the iCAN database. Each of the unique CDR's wasindividually synthesized and shuffled in the partially humanizedframework using the NUGE tool to generate a library with theoreticaldiversity of 3.2×10 9. The library was then cloned in the phagemidvector using the NUGE tool.

The Carterra SPR system was used to assess binding affinity and affinitydistribution for VHH-Fc variants. VHH-Fc demonstrate a range ofaffinities for TIGIT, with a low end of 12 nM K_(D) and a high end of1685 nM K_(D) (data not shown). Table 5A provides specific values forthe VHH-Fc clones for ELISA, Protein A (mg/ml), and K_(D) (nM). FIG. 7Aand FIG. 7B depict TIGIT affinity distribution for the VHH libraries,over the 20-4000 affinity threshold (FIG. 7A; monovalent KD) and the20-1000 affinity threshold (FIG. 7B; monovalent KD). Out of the 140 VHHbinders tested, 51 variants had affinity <100 nM, and 90 variants hadaffinity <200 nM. FIG. 8 shows data of CDR3 counts per length for the‘VHH ratio’ library, the ‘VHH shuffle library,’ and the ‘VHH hShufflelibrary.’ Table 5B shows number of TIGIT unique clones and TIGIT bindersfor the ‘VHH ratio’ library, the ‘VHH shuffle library,’ and the ‘VHHhShuffle library.’

TABLE 5A ProA K_(D) Clone ELISA Library (mg/m1) (nM) 31-1  5.7 VHHhShuffle 0.29 12 31-6  9.6 VHH hShuffle 0.29 14 31-26 5.1 VHH hShuffle0.31 19 30-30 8.0 VHH Shuffle 0.11 23 31-32 8.0 VHH hShuffle 0.25 2729-10 5.0 VHH Ratio 0.19 32 29-7  7.3 VHH Ratio 0.28 41 30-43 13.5 VHHShuffle 0.18 44 31-8  12.7 VHH hShuffle 0.29 45 31-56 11.7 VHH hShuffle0.26 46 30-52 4.2 VHH Shuffle 0.22 49 31-47 8.8 VHH hShuffle 0.23 5330-15 9.3 VHH Shuffle 0.26 55 30-54 5.5 VHH Shuffle 0.30 58 30-49 10.3VHH Shuffle 0.26 62 29-22 3.4 VHH Ratio 0.27 65 29-30 9.2 VHH Ratio 0.2865 31-35 5.7 VHH hShuffle 0.24 66 29-1  10.4 VHH Ratio 0.09 68 29-6  6.8VHH Ratio 0.29 69 31-34 6.0 VHH hShuffle 0.32 70 29-12 6.2 VHH Ratio0.23 70 30-1  5.4 VHH Shuffle 0.39 71 29-33 3.9 VHH Ratio 0.15 74 30-204.6 VHH Shuffle 0.19 74 31-20 6.6 VHH hShuffle 0.37 74 31-24 3.1 VHHhShuffle 0.15 75 30-14 9.9 VHH Shuffle 0.19 75 30-53 7.6 VHH Shuffle0.24 78 31-39 9.9 VHH hShuffle 0.32 78 29-18 10.9 VHH Ratio 0.19 7830-9  8.0 VHH Shuffle 0.40 79 29-34 8.6 VHH Ratio 0.21 80 −29-27  8.6VHH Ratio 0.18 82 29-20 5.9 VHH Ratio 0.26 83 30-55 6.0 VHH Shuffle 0.4185 30-39 6.1 VHH Shuffle 0.07 88 31-15 6.2 VHH hShuffle 0.32 88 29-214.3 VHH Ratio 0.23 88 29-37 5.3 VHH Ratio 0.26 89 29-40 6.6 VHH Ratio0.31 90 31-30 3.2 VHH hShuffle 0.33 93 31-10 12.3 VHH hShuffle 0.31 9429-3  13.6 VHH Ratio 0.11 94 30-57 5.2 VHH Shuffle 0.24 95 29-31 4.4 VHHRatio 0.18 96 31-27 8.1 VHH hShuffle 0.31 96 31-33 6.0 VHH hShuffle 0.3296 30-40 7.1 VHH Shuffle 0.21 99 31-18 4.1 VHH hShuffle 0.36 99 30-5 9.3 VHH Shuffle 0.05 100

TABLE 5B TIGIT unique clones and TIGIT binders Library Unique PhageVHH-Fc binders VHH Ratio 47 36 VHH Shuffle 58 45 VHH hShuffle 56 53

Thermostability and competition analysis of the VHH-Fc TIGIT clones isseen in FIG. 9 and Table 6. For the competition assays, 4 ug/mL TIGITwas immobilized and incubated with 0.05-100 nM VHH-Fc followed byincubation with 2 ug/mL biotin-CD155 and 1:5000 streptavidin-HRP.

TABLE 6 Thermostability of VHH-Fc TIGIT clones K_(D) Variant Library(nM) T_(m1) T_(m2) IC50 (nM) TIGIT-29-10 Ratio 32 72 87 17.65 TIGIT-29-7Ratio 41 82 90 9.24 TIGIT-30-30 Shuffle 23 76 87 5.67 TIGIT-30-43Shuffle 44 82 90 2.32 TIGIT-31-1 hShuffle 12 79 89 17.89 TIGIT-31-6hShuffle 14 77 87 4.00 TIGIT-31-26 hShuffle 19 79 89 8.20 TIGIT-31-32hShuffle 27 80 86 2.85 TIGIT-31-8 hShuffle 45 76 84 3.92 TIGIT-31-56hShuffle 46 74 83 1.52

CD47 VHH variants were also generated and analyzed. FIG. 10 shows theCD47 affinity distribution. Table 7 shows number of CD47 unique clonesand TIGIT binders for the ‘VHH ratio’ library, the ‘VHH shufflelibrary,’ and the ‘VHH hShuffle library.’ Table 8 shows the bindingaffinity of the CD47 VHH variants. As seen in Table 8, 8 CD47 VHHbinders had an affinity less than 100 nM to hCD47 and 6 CD47 VHH bindershad an affinity less than 100 nM to cCD47.

TABLE 7 VHH-Fc CD47 clones Library Unique Phage VHH-Fc binders VHH Ratio3 2 VHH Shuffle 1 1 VHH hShuffle 11 6

TABLE 8 VHH-Fc CD47 binding affinities hCD47 cCD47 Variant K_(D) (nM)K_(D) (nM) CD47-19-2 35 93 CD47-19-3 200 — CD47-20-1 49 105 CD47-21-1 2880 CD47-21-2 31 80 CD47-21-3 19 43 CD47-21-4 62 265 CD47-21-6 71 69CD47-21-10 38 35

Inhibition and thermostability analysis of the VHH-Fc CD47 clones isseen in FIG. 11 and Table 9. For the inhibition assays, 3 ug/mL of CD47was immobilized and incubated with 0.3-132 nM of VHH-Fc followed byincubation with 0.25 ug/mL biotin-SIRP alpha and 1:5000streptavidin-HRP.

TABLE 9 Thermostability of VHH-Fc CD47 clones K_(D) SIRPalpha Variant(nM) T_(m1) T_(m2) IC50 (nM) CD47-19-2 35 75 88 1.13 CD47-19-3 200 76 871.08 CD47-20-1 49 78 89 1.79 CD47-21-1 28 80 88 1.68 CD47-21-2 31 69 88— CD47-21-3 19 80 88 1.35 CD47-21-4 62 81 89 — CD47-21-6 71 79 88 —CD47-21-10 38 71 85 —

Example 5. VHH Libraries for GLP1R

A VHH library for GLP1R was developed similar to methods described inExample 4. Briefly, stable cell lines expressing GLP1R were generated,and target expression was confirmed by FACS. Cells expressing >80% ofthe target were then used for cell-based selections. Five rounds ofcell-based selections were carried out against cells stablyoverexpressing the target of interest. 10⁸ cells were used for eachround of selection. Before selection on target expressing cells, phagefrom each round was first depleted on 10⁸ CHO background cells.Stringency of selections was increased by increasing the number ofwashes in subsequent rounds of selections. The cells were then elutedfrom phage using trypsin, and the phage was amplified for the next roundof panning. A total of 1000 clones from round 4 and round 5 aresequenced by NGS to identify unique clones for reformatting as VHH-Fc.

53 out of the 156 unique GLP1R VHH Fc binders had a target cell meanfluorescence intensity (MFI) value that was 2-fold over parental cells.The data for variant GLP1R-43-77 is seen in FIGS. 12A-12B and Tables10-11. Table 11 shows flow cytometry data as detected with the RL1-Achannel.

TABLE 10 Panning summary VHH-Fc FACS binders (MFI values 2-fold LibraryUnique Phage over parental cells) VHH hShuffle 58 6 VHH Ratio/Shuffle 9847

TABLE 11 GLP1R-43-77 data Subset Name with Gating Path Count Median:RL1-A Sample E10.fcs/CHO-parent 11261 237 Sample E10.fcs/CHO-GLP1R 1368423439

Example 6. VHH Libraries for CRTH2R

A VHH library for CRTH2R was developed similar to methods described inExample 4. Briefly, stable cell lines expressing CRTH2R were generated,and target expression was confirmed by FACS. Cells expressing >80% ofthe target were then used for cell-based selections. Five rounds ofcell-based selections were carried out against cells stablyoverexpressing the target of interest. 10⁸ cells were used for eachround of selection. Before selection on target expressing cells, phagefrom each round was first depleted on 10⁸ CHO background cells.Stringency of selections was increased by increasing the number ofwashes in subsequent rounds of selections. The cells were then elutedfrom phage using trypsin, and the phage was amplified for the next roundof panning. A total of 1000 clones from round 4 and round 5 aresequenced by NGS to identify unique clones for reformatting as VHH-Fc.

26 binders out of the 175 unique CRTH2R VHH Fc binders had a target cellmean fluorescence intensity (MFI) value that was 2-fold over parentalcells. The data for variant CRTH2-41-51 is seen in FIGS. 13A-13B andTables 12-13. Table 13 shows flow cytometry data as detected with theRL1-A channel. Data for variant CRTH2-44-59 is seen in FIGS. 14A-14D.

TABLE 12 Panning summary VHH-Fc FACS binders (MFI values 2 fold LibraryUnique Phage over parental cells) VHH hShuffle 99 16 VHH Ratio/Shuffle76 10

TABLE 13 CRTH2-41-51 data Sample Name Subset Name Count Median: RL1-ASample C7.fcs CRTH2R cells 8663 7441 Sample E10.fcs Parent Cells 115892120

Example 7. Identification of IgGs for CRTH2R

Cell binding of anti-CRTH2R antibodies was determined by testing on CHOCRTH2R-positive cells (GFP+) and parental CHO cells (GFP−), comparingparental negative and target positive cells to rule out false-positives.Antibodies as listed in Table 14A were titrated starting at 100 nM (15ug/mL) with 3-fold titrations, for a total of 8 points. Heavy and lightchain sequences for CRTH2R IgG antibodies are shown in Table 14B.Binding as detected by mean fluorescence intensity (MFI) byconcentration is shown in FIGS. 15A-15E. An exemplary gated dot plot andAPC histogram at 100 nM with CRTH2-27 is shown in FIGS. 16A-6B. Twoantibodies (gPCR-51 and gPCR-52) were used as a positive control.Binding profiles of the two positive controls are shown in FIGS.17A-17B.

TABLE 14A CRTH2R antibody variable heavy and light chain sequences SEQCRTH2R ID NO Antibody Heavy Chain 1 CRTH2-74QVQLVESGGGVVQPGRSLRLSCAASGFSFSEYGIHWVRQAPGKGLEWVAVISYEGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARANQHFGPVAGGATPSEEPGSQLTRAELGWDAPPGQESLADELLQLGlEHGYHYYGMDVWGQG TLVTVSS 2 CRTH2-24QVQLVQSGAEVKKPGSSVKVSCKASGGSFSNYGISWVRQAPGQGLEWMGGIIPLIGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFTLGPQSIGPLGEVVPADDAFDIWGQGTLVTVSS 3 CRTH2-28QVQLVQSGAEVKKPGSSVNVSCKASGGTFSDYAFSWVRQAPGQGLEWMGAIIPFFGTVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFATGTGGPEDDLYPQGELNDGYRIEVVPADDAFDIWGQGTLVTVSS 4 CRTH2-39QVQLVQSGAEVKKPGSSVKVSCKASVDTFSRYSISWVRQAPGQGLEWMGGIIPVFDTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFGVILGGTAVGTNNGSANEVVPADDAFDIWGQGTLVTVSS 5 CRTH2-19QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHAINWVRQAPGQGLEWMGRIIPIVGTTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFDYFGLTLTGDRNDDEVVPADDAFDIWGQGTLVTVSS 6 CRTH2-9QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFWLGDQSTGSLIGAEVVPADDAFDIWGQGTLVTVSS 7 CRTH2-8QVQLVQSGAEVKKPGSSVKVSCKASGGTFTDYAISWVRQAPGQGLEWMGGIIPFFGSPNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFAAGLEGTITEVFDEEGHQGGTEVVPADDAFDIWGQGTLVTVSS 8 CRTH2-27QVQLVESGGGVVQPGRSLRLSCAASGFTFDNYGMHWVRQAPGKGLEWVAVISYEGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDMYYDFGSIYGEDVVGELPEVVPADDAFDIWGQGTLVTVSS 9 CRTH2-45QVQLVESGGGVVQPGRSLRLSCAASGFTFSHYAMHWVRQAPGKGLEWVADISHEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGRGSLPRPKGGPTSGGGFSTNIGYGFVVQSYDSSEDSGGAFDIWGQGTLVTVSS 10 CRTH2-35QVQLVQSGAEVKKPGSSVKVSCKASGGTFRSYAISWVRQAPGQGLEWMGGIIPISGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFTRIFGNYQIYFGHFGYHYYGMDVWGQGTLVTVSS 11 CRTH2-50QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYALSWVRKAPGQGLEWMGGTIPIFGTVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFTRVIGQPSPAVPSRGYIYHGYHYYGMDVWGQGTLVTVSS 12 CRTH2-66QVQLVESGGGVVQPGRSLRLSCAASGFDFSGYGMHWVRQAPGKGLEWVAVI SYEGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRELECEEWTIEVHGQEFAVHQDRGGVFSRGPCVDPRGVAGSFDVWGQGTLVTVSS 13 CRTH2-57QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVRQAPGQGLEWMGGIIPLFGTTDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFVKIQGAPVSTPVPGFGTTGYHYYGMDVWGQGTLVTVSS 14 CRTH2-32QVQLVESGGGVVQPGRSLRLSCAASGFTFSKHGMHWVRQAPGKGLEWVAFISYEGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDMYYDFHYSTVGATYYYYLGSETEVVPADDAFDIWGQGTLVTVSS 15 CRTH2-15QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAIDWVRQAPGQGLEWMGGIIPLFGSPNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFFLYEGTSSSWLHVGHARYGYHYYGMDVWGQGTLVTVSS 16 CRTH2-25QVQLVQSGAEVKKPGSSVKVSCKASGGSFRSYGISWVRQAPGQGLEWMGRIIPLFGTPDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFEDVDEGSLYLDMGRTFEVVPADDAFDIWGQGTLVTVSS 17 CRTH2-42QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYAMHWVRQAPGKGLEWVAVISYEGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRELECEEWTVLQYGKFHMRWAESGEGSLSRGPCVDPRGVAGSFDVWGQGTLVTVSS 18 CRTH2-55QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYDMHWVRQAPGKGLEWVAVISYEGSEKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQASTEGDFGLEEVTGEGVDDRADLVGDAFDVWGQGTLVTVSS 19 CRTH2-60QVQLVQSGAEVKKPGSSVKVSCKASGGTFKNYAINWVRQAPGQGLEWMGAIIPKFGAANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFSAVRGLAFGYGYRIGGYHYYGMDVWGQGTLVTVSS 20 CRTH2-70QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNHAIIWVRQAPGQGLEWMGGIIPIFGTPSYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMYYDFDVISAGVVGAGNPEVVPADDAFDIWGQGTLVTVSS 21 CRTH2-48-9EVQLLESGGGLVQPGGSLRLSCAASGFSFSTHAMSWVRQAPGKGLEWVSTIGGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAHGDSSSWYFSYYYMDVWGQGTLVTVSS 22 CRTH2-41-51EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAGISGSGGDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWG QGTLVTVSS 23CRTH2-44-6 EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISMSGDDTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLRGRGDVS EYEYDWGQGTLVTVSSSEQ CRTH2R ID NO Antibody Light Chain 24 CRTH2-74QSVLTQPPSVSAAPGQKVTISCSGSTSNIGKNYVSWYQQLPGTAPKLLIYDDDERPSGIPDRFSGSMSGTSATLGITGLQTGDEADYYCEAWDADLSGAVFGGGTKLTVL 25 CRTH2-24QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNFVSWYQQLPGTAPKLLIYDNIQRPSGIPDRFSGSK SGTSATLGITGLQTGDEADYYCGTWDTSLSAVVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 26 CRTH2-28QSVLTQPPSVSAAPGQKVTISCSGSISNIGKNYVSWYQQLPGTAPKLLIYDDHKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDRGLSAAVFGGGTKLTVL 27 CRTH2-39QSVLTQPPSVSAAPGQKVTISCSGSSSNIGDNDVSWYQQLPGTAPKLLIYDDDKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCASWDTSLSGGYVFGGGTKLTVL 28 CRTH2-19QSALTQPASVSGSPGQSITISCTGTSSDVGGYDYVTWYQQHPGKAPKLMIYDVDTRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTSTSYVFGGGTKLTVL 29 CRTH2-9*QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIYENDERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDTRLSAVVFGGGTKLTVL 30 CRTH2-8QSVLTQPPSVSAAPGQKVTISCSGSSSNIGKNYVSWYQQLPGTAPKLLIYDNNQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLTSVVFGGGTKLTVL 31 CRTH2-27QSALTQPASVSGSPGQSITISCTGTSNDVGAYNFVSWYQQHPGKAPKLMIYDISNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTRSNTRVFGGGTKLTVL 32 CRTH2-45QSVLTQPPSVSAAPGQKVTISCSGTSSNIENNYVSWYQQLPGTAPKLLIYDNVKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNTVSAPWVFGGGTKLTVL 33 CRTH2-35QSALTQPASVSGSPGQSITISCTGTSSDIGGYEFVSWYQQHPGKAPKLMIYGVSRRPS GVSNRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTPYVFGGGTKLTVL 34 CRTH2-50QSALTQPASVSGSPGQSITISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIYDVSNRPQGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSNTYVVFGGGTKLTVL 35 CRTH2-66EIVMTQSPATLSVSPGERATLSCRASQGVGSNLAWYQQKPGQAPRLLIYRTSIRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYYSWPPLTFGGGTKVEIK 36 CRTH2-57QSVLTQPPSVSAAPGQKVTISCSGSSSNIEDNYVSWYQQLPGTAPKLLIYDNFKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSAALFGGGTKLTVL 37 CRTH2-32QSALTQPASVSGSPGQSITISCTGTSSGVGGYDYVSWYQQHPGKAPKLMIYDDNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTGSSTLYVFGGGTKLTVL 38 CRTH2-15QSVLTQPPSVSAAPGQKVTISCSGSGSNIGSNYVSWYQQLPGTAPKLLIYDNIRRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCAAWDTRLSAGVFGGGTKLTVL 39 CRTH2-25DIQMTQSPSSLSASVGDRVTITCRASQGISTYLNWYQQKPGKAPKLLIYATSSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP-WTFGGGTKVEIK 40 CRTH2-42QSALTQPASVSGSPGQSITISCTGTSSDVGGYRYVSWYQQHPGKAPKLMIYNVNYRP SGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYRSSSTLGVFGGGTKLTVL 41 CRTH2-55QSVLTQPPSVSAAPGQKVTISCSGSSSNIGDNFVSWYQQLPGTAPKLLIYDDDERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGAWDRSLSAVVFGGGTKLTVL 42 CRTH2-60QSVLTQPPSVSAAPGQKVTISCSGSTSNIGINYVSWYQQLPGTAPKLLIYENRKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDASLKNLVFGGGTKLTVL 43 CRTH2-70QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNFVSWYQQLPGTAPKLLIYDNEKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDERQTDESYVFGGGTKLTV L 44 CRTH2-9AQSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIYENDERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDTRLSAVVFGGETKLT 45 CRTH2-48-9DIQMTQSPSSLSASVGDRVTITCRASQSISDYVNWYQQKPGKAPKLLIYGASILQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFTTPWTFGGGTKVEIK

TABLE 14B Variably Heavy Chain CDR3 Sequences SEQ CRTH2R ID NO AntibodyCDRH3 46 CRTH2-74 CARANQHFGPVAGGATPSEEPGSQLTRAELGWDAPPGQESLADELLQLGTEHGYHHYYGMDVW 47 CRTH2-24CARDMYYDFTLGPQSIGPLGEVVPADDAFDIW 48 CRTH2-28CARDMYYDFATGTGGPEDDLYPQGELNDGYRIEV VPADDAFDIW 49 CRTH2-39CARDMYYDFGVILGGTAVGTNNGSANEVVPADDA FDIW 50 CRTH2-19CARDMYYDFDYFGLTLTGDRNDDEVVPADDAFDI W 51 CRTH2-9CARDMYYDFWLGDQSTGSLIGAEVVPADDAFDIW 52 CRTH2-8CARDMYYDFAAGLEGTITEVFDEEGHQGGTEVVP ADDAFDIW 53 CRTH2-27CARDMYYDFGSIYGEDVVGELPEVVPADDAFDIW 54 CRTH2-45CARDGRGSLPRPKGGPTSGGGFSTNIGYGFVVQS YDSSEDSGGAFDIW 55 CRTH2-35CARANQHFTRIFGNYQIYFGHFGYHYYGMDVW 56 CRTH2-50CARANQHFTRVIGQPSPAVPSRGYIYHGYHYY GMDVW 57 CRTH2-66CARDLRELECEEWTIEVHGQEFAVHQDRGGVFSR GPCVDPRGVAGGSFDVW 58 CRTH2-57CARANQHFVKIQGAPVSTPVPGFGTTGYHYYGM DVW 59 CRTH2-32CARDMYYDFHYSTVGATYYYYLGSETEVVPADDA FDIW 60 CRTH2-15CARANQHFFLYEGTSSSWLHVGHARYGYHYYYGM DVW 61 CRTH2-25CARDMYYDFEDVDEGSLYLDMGRTFEVVPADDAF DIW 62 CRTH2-42CARDLRELECEEWTVLQYGKFHMRWAESGEGSLS RGPCVDPRGVAGSFDVW 63 CRTH2-55CAKHMSMQASTEGDFGLEEVTGEGVDDRADLVGD AFDVM 64 CRTH2-60CARANQHFSAVRGLAFGYGYRIGGYHYYGMDVW 65 CRTH2-70CARDMYYDFDVISAGVVGAGNPEVVPADDAFDIW 66 CRTH2-74CARDMYYDFDVISAGVVGAGNPEVVPADDAFDIW

In subsequent examples, five antibodies were shown to have functionaleffects in cAMP assays: CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, andCRTH2-42. The binding curves of these antibodies are compared in FIGS.18A-18B.

Example 8. Antagonist Activity Using cAMP Assay

A library of CRTH2R IgG antibodies were assayed to determine antagonistfunction in PGD2-induced cAMP signals. Briefly, cells were pre-incubatedwith IgG (titration 1:3) for 1 hour at room temperature. Subsequently,cells were stimulated with PGD2 (0.59 nM) for 30 min at 37° C. in thepresence of forskolin, since CRTH2R is Gα_(i) coupled.

Effect of antibody on detected signal in relative light units (rlu) wasdetermined (data not shown). At the highest concentration tested (300nM), some of the CRTH2R IgGs caused an upward deflection of the signal,indicating inhibition of the cAMP signal induced by PGD2 stimulation.For comparison, bar charts showing the ratio of IgG treated versuscontrol treated for the three highest IgG concentrations tested areshown in FIG. 19A. Antibodies depicted in FIG. 19B show CRTH2R IgGantibodies which resulted in more than a 20% antagonist activity at 33nM, specifically CRTH2-74, CRTH2-24, CRTH2-28, CRTH2-19, CRTH2-45,CRTH2-9, CRTH2-8, CRTH2-15, CRTH2-42, CRTH2-60, and CRTH2-70.

Example 9. Allosteric Modulation of PGD2-Induced cAMP Signal

CRTH2R IgG antibodies were assayed for allosteric activity. Allostericmodulation was determined by assaying CRTH2R IgG antibodies inPGD2-induced cAMP signal. Briefly, cells were re-incubated with no IgGantibody or 100 nM CRTH2R IgG antibody. Subsequently, cells werestimulated with PGD2 at various concentrations in the presence offorskolin followed by assay for cAMP activity.

Results of the cAMP assays is seen in FIG. 20. A right-ward shift thePGD2 dose response curve (and increase in IC50 value) indicates anegative allosteric effect. As shown in FIG. 20, five of the CRTH2R IgG(CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, and CRTH2-42) caused an IC50fold difference of >2.0 compared with PGD2 alone, suggesting they arenegative allosteric modulators.

Example 10. Agonist Activity of PGD2-Induced cAMP Signal

CRTH2R IgG antibodies were assayed for agonist function. Agonistactivity was determined by assaying CRTH2R IgG antibodies described inExample 7 in PGD2-induced cAMP signal.

Briefly, cells were treated with PGD2 or CRTH2R IgG antibodies both inthe presence of forskolin. The CRTH2R IgG antibodies included CRTH2-74,CRTH2-24, CRTH2-28, CRTH2-39, CRTH2-19, CRTH2-9, CRTH2-8, CRTH2-27,CRTH2-45, CRTH2-35, CRTH2-50, CRTH2-66, CRTH2-57, CRTH2-32, CRTH2-15,CRTH2-25, CRTH2-42, CRTH2-55, CRTH2-60, and CRTH2-70. Treatmentstimulations were performed for 30 min at 37° C. cAMP assays were thenperformed (data not shown).

Example 11. Control Experiments Showing Allosteric Modulators

Allosteric modulation was determined for a known CRTH2R antagonist(small molecule OC000459) and two control antibodies. Experiments wereperformed similar to those described in Example 9. Briefly, cells weretreated with OC000459, comparator CRTH2R AB51 antibody, or comparatorCRTH2R AB52 antibody. Cells were then stimulated with PGD2 in thepresence of forskolin.

Results are shown in FIGS. 21A-21C. OC000459 causes a strong right-wardshift of the curve and a 459-fold increase in the IC50 value (FIG. 21A).Incubation with CRTH2R AB51 caused no change in IC50 value (FIG. 21B).Incubation with the comparator antibody #52 caused a 3.5-fold decreasein the IC50 value, indicating it is a positive allosteric modulator,i.e. it has agonistic effects (FIG. 21C).

Example 12. CRTH2R β-arrestin Recruitment Assay for AntagonistModulation

Antagonist modulation by nine CRTH2R IgG antibodies was determined. Thenine CRTH2R IgG antibodies included CRTH2-9, CRTH2-27, CRTH2-50,CRTH2-32, CRTH2-42, CRTH2-74, CRTH2-55, CRTH2-28, and CRTH2-39. Theantagonist function of these nine antibodies as compared to OC000459 wasdetermined using a PGD2-induced β-arrestin recruitment. Results,including a positive control using small molecule OC 000459, are shownin FIGS. 22A-22D.

Example 13. CRTH2R β-arrestin Recruitment Assay for AllostericModulation

Allosteric modulation by nine CRTH2R IgGs were determined. The nineCRTH2R IgGs included CRTH2-9, CRTH2-27, CRTH2-50, CRTH2-32, CRTH2-42,CRTH2-74, CRTH2-55, CRTH2-28, and CRTH2-39. The allosteric modulation ofthese nine antibodies as compared to OC000459 was determined using aPGD2-induced β-arrestin recruitment.

Briefly, cells were pre-incubated with IgG (100 nM) for 1 hour at roomtemperature followed by PGD2 stimulation for 90 min at 37° C. Data wasnormalized against the first data point (lowest PGD2 and zero Ab) ineach graph.

Example 14. Hyperimmune Immunoglobulin Library

A hyperimmune immunoglobulin (IgG) library was created using similarmethods as described in Example 4. Briefly, the hyperimmune IgG librarywas generated from analysis of databases of human naïve and memoryB-cell receptor sequences consisting of more than 37 million unique IgHsequences from each of 3 healthy donors. More than two million CDRH3sequences were gathered from the analysis and individually constructedusing methods similar to Examples 1-3. Any duplicate CDRH3's andpotential liability motifs that frequently pose problems in developmentwere removed during the library synthesis step. These CDRH3 sequencediversities were then combinatorially assembled and incorporated ontothe DP47 human framework to construct a highly functional antibody Fablibrary with 1×10¹⁰ size. A schematic of the design can be seen in FIG.24.

The heavy chain CDR length distribution of the hyperimmune antibodylibraries were assessed by next generation sequencing (NGS). The data ofCDR length distribution is shown in FIGS. 25A-25B. Generally, selectionof soluble protein targets undergo five rounds of selection involving aPBST wash three times in Round 1, a PBST wash five times in Round 2, aPBST wash seven times in Round 3, a PBST wash nine times in Round 4, anda PBST wash twelve times in Round 5. A non-fat milk block was used. SeeFIG. 26.

For human TIGIT (hTIGIT), 1 uM biotinylated antigen was mixed with 300ul Dynabead M-280 at 10 mg/mL to generate a concentration of 100 pmolper 100 ul. The details of the various rounds of selection are seen inTable 15.

TABLE 15 Protein panning selection Round Washes Antigen AmountConcentration Manual 1 3 100 pmol 1 uM 2 6 20 pmol 200 nM 3 9 10 pmol100 nM 4 12 5 pmol 50 nM 5 12 5 pmol 50 nM Kingfisher (KF) 1 2 100 pmol1 uM 2 4 20 pmol 200 nM 3 6 10 pmol 100 nM 4 8 5 pmol 50 nM 5 8 5 pmol50 nM

After various rounds of selection, hTIGIT IgGs were analyzed. Data isseen in FIGS. 27A-27F and Table 16. FIGS. 27A-27D show ELISA data fromRound 3 and Round 4. FIGS. 27E-27F show data of CDRH3 length, yield(ug), and K_(D) (nM) for the hTIGIT IgGs analyzed.

TABLE 16 Protein panning data KF Round Target Antigen Washes WashesTiter KF liter 1 hTIGIT 100 pmol 3 — 4.40E+06 — 2 hTIGIT 50 pmol 5 44.40E+07 6.80E+06 3 hTIGIT 20 pmol 7 4 6.00E+08 2.80E+09 4 hTIGIT 10pmol 9 5 5.00E+08 6.00E+08 5 hTIGIT 10 pmol — — — —

Seventeen non-identical hTIGIT immunoglobulins were identified withmonovalent affinity ranging from 16 nM to over 300 nM. Most of theseimmunoglobulins expressed well and produced over 20 ug purified proteinat 1 ml expression volume. Sequences for hTIGIT immunoglobulins are seenin Table 17.

TABLE 17 TIGIT sequences SEQ ID NO: IgG Amino Acid Sequence CDRH3 67TIGIT-55-01 CARVAGSSGWAFDYW 68 TIGIT-55-02 CATLRLYSSGGGIDYW 69TIGIT-55-03 CARIVGATTRTYYYYGMDVW 70 TIGIT-55-04 CARVRNRASDIW 71TIGIT-55-05 CARAPYSSSSWFDYW 72 TIGIT-55-06 CARNSYGPPRSFGMDVW 73TIGIT-55-07 CARTPYRSGWADYW 74 TIGIT-55-08 CTRSWYYYYGMDVW 75 TIGIT-55-09CARGYGGYGYW 76 TIGIT-55-10 CAKAGDYDYYFDYW 77 TIGIT-55-11 CASVKRWGYYFNWW78 TIGIT-55-12  CARVRVGAYDAFDIW 79 TIGIT-55-13  CARNSGWFMPFDYW 80TIGIT-55-14  CARRGSGWYIDSW 81 TIGIT-55-15  CARREGDYMGPNWFDPW 82TIGIT-55-16  CASIRERRFDFW 83 TIGIT-55-17  CARHSLTPYNFWSGYYSRSFDIWVariable Heavy Chain 84 TIGIT-55-01EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYGMSWVRQAPGKGLEWVSSISGSGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVAGSSGW AFDYWGQGTLVTVSS 85TIGIT-55-02 EVQLLESGGGLVQPGGSLRLSCAASGLTFSNYAMTWVRQAPGKGLEWVSGISRSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATLRLYSSGG GIDYWGQGTLVTVSS 86TIGIT-55-03 EVQLLESGGGLVQPGGSLRLSCAASGFTFHNYAMTWVRQAPGKGLEWVSAITGSGTSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIVGATTRTYYYYGMDVWGQGTLVTVSS 87 TIGIT-55-04EVQLLESGGGLVQPGGSLRLSCAASGFRFGNYAMSWVRQAPGKGLEWVSAITGSGGNTFYADSVKGRFTISRDNSKNTLYLQINSLRAEDTAVYYCARVRNRASDI WGQGTLVTVSS 88TIGIT-55-05 EVQLLESGGGLVQPGGSLRLSCAASGFVFSSYAMNWVRQAPGKGLEWVSTVSGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAPYSSSS WFDYWGQGTLVTVSS 89TIGIT-55-06 EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYTMNWVRQAPGKGLEWVSGISGSGGGAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNSYGPPRS FGMDVWGQGTLVTVSS90 TIGIT-55-07 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMTWVRQAPGKGLEWVSAISGRGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTPYRSGW ADYWGQGTLVTVSS 91TIGIT-55-08 EVQLLESGGGLVQPGGSLRLSCAASGFMFSDYAMSWVRQAPGKGLEWVSGISGSGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRSWYYYY GMDVWGQGTLVTVSS 92TIGIT-55-09 EVQLLESGGGLVQPGGSLRLSCAASGFAFRSYAMGWVRQAPGKGLEWVSTISGGGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGGY GYWGQGTLVTVSS 93TIGIT-55-10 EVQLLESGGGLVQPGGSLRLSCAASGFTFSKSAMSWVRQAPGKGLEWVSAISGSGGLTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGDYDYY FDYWGQGTLVTVSS 94TIGIT-55-11 EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYGMSWVRQAPGKGLEWVSSISGSGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASVKRWGYY FNWWGQGTLVTVSS 95TIGIT-55-12 EVQLLESGGGLVQPGGSLRLSCAASGFTLSSYAMAWVRQAPGKGLEWVSTLSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRVGAY DAFDIWGQGTLVTVSS 96TIGIT-55-13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMNWVRQAPGKGLEWVSTISGSGGSTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNSGWFMPF DYWGQGTLVTVSS 97TIGIT-55-14 EVQLLESGGGLVQPGGSLRLSCAASGFMFSRYAMSWVRQAPGKGLEWVSSISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSGWYI DSWGQGTLVTVSS 98TIGIT-55-15 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMGWVRQAPGKGLEWVSTISGSGSRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREGDYMG PNWFDPWGQGTLVTVSS99 TIGIT-55-16 EVQLLESGGGLVQPGGSLRLSCAASGFAFSSYAMGWVRQAPGKGLEWVSAITSSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASIRERRFDF WGQGTLVTVSS 100TIGIT-55-17 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVSGISGSGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSLTPYNFWSGYYSRSFDIWGQGTLVTVSS Variable Light Chain 101 TIGIT-55-01DIQMTQSPSSLSASVGDRVTITCRASQAISNYLNWYQQKPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQESYSTPFTFGGGTKVEIK 102 TIGIT-55-02DIQMTQSPSSLSASVGDRVTITCRASQYISTYLNWYQQKPGKAPKLLIYAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYITPLTFGGGTKVEIK 103 TIGIT-55-03DIQMTQSPSSLSASVGDRVTITCRASQYISSYLNWYQQKPGKAPKLLIYGAFSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTFGGGTKVEIK 104 TIGIT-55-04DIQMTQSPSSLSASVGDRVTITCRASQTIITYLNWYQQKPGKAPKLLIYAASNLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSLPWTFGGGTKVEIK 105 TIGIT-55-05DIQMTQSPSSLSASVGDRVTITCRASQSVRSYLNWYQQKPGKAPKLLIYTATSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGLPRTFGGGTKVEIK 106 TIGIT-55-06DIQMTQSPSSLSASVGDRVTITCRASQSISKYLNWYQQKPGKAPKLLIYGASSLRGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRPPLTFGGGTKVEIK 107 TIGIT-55-07DIQMTQSPSSLSASVGDRVTITCRASQNIKTYLNWYQQKPGKAPKLLIYAASSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSIPQTFGGGTKVEIK 108 TIGIT-55-08DIQMTQSPSSLSASVGDRVTITCRAGQSIRSYLNWYQQKPGKAPKLLIYASSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLLTFGGGTKVEIK 109 TIGIT-55-09DIQMTQSPSSLSASVGDRVTITCRASQSIRRYLNWYQQKPGKAPKLLIYAASTLQIGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSSPYTFGGGTKVEIK 110 TIGIT-55-10DIQMTQSPSSLSASVGDRVTITCRTSQSIRRYLNWYQQKPGKAPKLLIYRASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTLRTFGGGTKVEIK 111 TIGIT-55-11DIQMTQSPSSLSASVGDRVTITCRASQNINYYLNWYQQKPGKAPKLLIYGASSLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTGGGTKVEIK 112 TIGIT-55-12DIQMTQSPYSLSASVGDRVTITCRASQSIRRYLNWYQQKPGKAPKLLTYRASTLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSSPFTFGGGTKVEIK 113 TIGIT-55-13DIQMTQ SPSSLSASVGDRVTITCRTSQSISTYLNWYQQKPGKAPKLLIYATSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPLTFGGGTKVEIK 114 TIGIT-55-14DIQMTQSPSSLSASVGDRVTITCRASQSVSRYLNWYQQKPGKAPKLLIYGSSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQESYSTPFTFGGGTKVEIK 115 TIGIT-55-15DIQMTQSPSSLSASVGDRVTITCRASQAISRNLNWYQQKPGKAPKLLIYGASNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPVTFGGGTKVEIK 116 TIGIT-55-16DIQMTQSPSSLSASVGDRVTITCRASQRISTYLNWYQQKPGKAPKLLIYGTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIIPWTFGGGTKVEIK 117 TIGIT-55-17DIQMTQSPSSLSASVGDRVTITCRASQSISSYVNWYQQKPGKAPKLLIYGASRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTFGGGTKVEIK

Identification of human CD3 epsilon (hCD3) and cyno CD3 epsilon (cCD3)immunoglobulins was performed. The details of the various rounds ofselection are seen in Table 18.

TABLE 18 Protein panning selection Round Washes Concentration Manual 1 3500 nM hCD3 and 500 nM cCD3 2 6 100 nM hCD3 3 9 50 nM hCD3 4 12 50 nMhCD3 5 12 10 nM hCD3 Kingfisher (KF) 1 2 500 nM hCD3 and 500 nM cCD3 2 4100 nM hCD3 3 6 50 nM hCD3 4 8 50 nM hCD3 5 8 10 nM hCD3

After various rounds of selection, CD3 epsilon (CD3E) IgGs wereanalyzed. Data is seen in FIGS. 28A-28L and Tables 19A-19B. FIGS.28A-28F show ELISA data from Round 4 and Round 5. FIGS. 28G-28L showdata of cross-reactivity of human CD3 epsilon and cyno CD3 epsilonimmunoglobulins.

TABLE 19A Protein panning data KF Round Target Antigen Washes WashesTiter KF Titer 1 hCD3/ 100 pmol 3 — 2.40E+06 — cCD3 2 hCD3 50 pmol 5 41.20E+08 1.80E+06 3 cCD3 20 pmol 7 4 8.00E+06 2.40E+07 4 hCD3 10 pmol 95 1.00E+07 2.10E+06 5 cCD3 10 pmol 12 7 1.50E+07 1.00E+08

TABLE 19B Output Round Target Antigen Washes Titer 1 hCD3 5 ug 49.00E+04 2 cCD3 5 ug 5 1.40E+05 3 hCD3 2.5 ug 6 3.00E+06 4 cCD3 2.5 ug 74.00E+06 5 hCD3 2.5 ug 8 2.20E+07

Nineteen non-identical hCD3 epsilon and cyno CD3 epsilon immunoglobulinswere identified including five that are human/cyno CD3 epsiloncross-reactive immunoglobulins. One of the human/cyno CD3 epsiloncross-reactive antibody, CD3-56-05 binds to human and cyno CD3 epsilonwith 67 and 107 nM affinity, respectively. Sequences for hCD3 epsilonand cCD3 epsilon immunoglobulins are seen in Table 20.

TABLE 20 CD3 epsilon sequences SEQ ID NO: IgG Amino Acid SequenceVariable Heavy Chain CDR1 (CDRH1) 118 CD3-138-6 GYTFTSNMH 119 CD3-56-5FTFSSYAMN 120 CD3-155-03 FTFSSYAIN Variable Heavy Chain CDR2 (CDRH2) 121CD3-138-6 VASISSYYGYTYYA 122 CD3-56-5 VSAVSGSGGRTYYA 123 CD3-155-03 VSALSGSGGSTYYA Variable Heavy Chain CDR3 (CDRH3) 124 CD3-138-6GGNYYNLWTGYYPLAY 125 CD3-56-5 ARERATTLDY 126 CD3-155-03  ARRSAQLGDY 127CD3-56-5A CARERATTLDYW 128 CD3-56-11  CARDSLTTRGYYYYMDVWVariable Light Chain CDR1 (CDRL1) 129 CD3-138-6 RASQDISTYLN 130 CD3-56-5RASQTIYSHLN 131 CD3-155-03 RASQSISSFLN Variable Light Chain CDR2 (CDRL2)132 CD3-138-6 YTDRLQT 133 CD3-56-5 VASRLQS 134 CD3-155-03  AAPSLQSVariable Light Chain CDR3 (CDRL3) 135 CD3-138-6 QQGGALPFT 136 CD3-56-5QQSFSTSWT 137 CD3-155-03 QQSFRTPFT Variable Heavy Chain 138 CD3-56-5EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAVSGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERATTLDYW GQGTLVTVSS 139CD3-56-11 EVQLLESGGGLVQPGGSLRLSCAASGFRFSTYAMNWVRQAPGKGLEWVSGISGSGGSKYHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSLTTRGYYY YMDVWGQGTLVTVSS140 CD3-138-6 EVQLLESGGGLVQPGGSLRLSCAASGGYTFTSNMHWVRQAPGKGLEWVASISSYYGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGNYYNLWTGY YPLAYWGQGTLVTVSS141 CD3-155-9 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYALNWVRQAPGKGLEWVSAVTGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRATTLDYW GQGTLVTVSSVariable Light Chain 142 CD3-56-5DIQMTQSPSSLSASVGDRVTITCRASQTIYSHLNWYQQKPGKAPKLLIYVASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSTSWTFGGGTKVEIK 143 CD3-56-11DIQMTQSQSSLSASVGDRVTITCRASQSIRTSLNWYQQPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLYSFGGGTKVEIK 144 CD3-138-6DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKLLIYYTDRLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGGALPFTFGQGTKVEIK 145 CD3-155-9DIQMTQSPSSLSASVGDRVTITCRTSQSISTYLNWYQQKPGKAPKLLIYTASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGGGTKVEIK

A CRTH2R hyperimmune immunoglobulin library was generated. Briefly, fiverounds of cell-based selections were carried out against cells stablyoverexpressing the target of interest. 10⁸ cells were used for eachround of selection. Before selection on target expressing cells, phagefrom each round was first depleted on 10⁸ CHO background cells.Stringency of selections was increased by increasing the number ofwashes in subsequent rounds of selections. The cells were then elutedfrom phage using trypsin, and the phage gets amplified for the nextround of panning.

CRTH2R immunoglobulins were assessed for binding affinity and allostericmodulator function of PGD2-induced cAMP. As seen in FIGS. 30A-30F, threespecific CRTH2R immunoglobulins were identified with sub nanomolar tosingle digit nanomolar cell binding affinities to hCRTH2R and hadinhibitory activities in the allosteric cAMP assay. The sequences forthe three CRTH2R immunoglobulins CRTH2-48-3, CRTH2-48-21, andCRTH2-48-27 are seen in Table 21.

TABLE 21 CRTH2R sequences SEQ ID NO: IgG Amino Acid SequenceVariable Heavy Chain 146 CRTH2-48-3EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDD RYDWGQGTQVTVSS 147CRTH2-48-21 EVQLVESGGGLVQAGGSLRLSCAASGSFFSINAMGWFRQAPGKEREFVAGITRSGVSTSYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAHRIVVGGTS VGDWRWGQGTQVTVSS148 CRTH2-48-27 EVQLVESGGGLVQAGGSLRLSCAASGSIFHINAMGWFRQAPGKEREGVAAINNFGTTKYADSVKGRFTISANNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDD RYDWGQGTLVTVSSVariable Light Chain 149 CRTH2-48-3DIQMTQSPSSLSASVGDRVTITCRASQSISSDLNWYQQKPGKAPKLLIYFASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPLTFGGGTKVEIKR 150 CRTH2-48-21DIQMTQSPSSLSASVGDRVTITCRTSQSISNYLNWYQQKPGKAPKLLIYATSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLLTFGGGTKVEIKR 151 CRTH2-48-27DIQMTQSPSSLSASVGDRVTITCRASQSISRYLHWYQQKPGKAPKLLIYGASRLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCRQSYSTPWTFGGGTKVEIKR

Example 15. Hyperimmune Immunoglobulin Library for A2A Receptor

A hyperimmune immunoglobulin (IgG) library was created using similarmethods as described in Examples 4 and 14. Briefly, the hyperimmune IgGlibrary was generated from analysis of databases of human naïve andmemory B-cell receptor sequences consisting of more than 37 millionunique IgH sequences from each of 3 healthy donors. More than twomillion CDRH3 sequences were gathered from the analysis and individuallyconstructed using methods similar to Examples 1-3. The CDRH3 sequenceswere incorporated into the VHH hShuffle library described in Example 4.The final library diversity was determined to be 1.3×10¹⁰.

73 out of 88 unique clones had a target cell MFI values 2 fold overparental cells. 15 out of 88 unique Clones with target cell MFI values20 fold over parental cells. Data for adenosine A2A receptor variantA2AR-90-007 is seen in FIGS. 31A-31B.

This Example shows generation of a VHH library for the A2AR with highaffinity and K_(D) values in the sub-nanomolar range.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1. An antibody or antibody fragment comprising a CDRH1 comprising anamino acid sequence at least about 90% identical to that set forth inSEQ ID NOs: 152 or 155, a CDRH2 comprising an amino acid sequence atleast about 90% identical to that set forth in SEQ ID NOs: 153 or 156,and a CDRH3 comprising an amino acid sequence at least about 90%identical to that set forth in SEQ ID NOs: 154 or
 157. 2. The antibodyor antibody fragment of claim 1, further comprising a CDRL1 comprisingan amino acid sequence at least about 90% identical to that set forth inSEQ ID NOs: 158 or 161, a CDRL2 comprising an amino acid sequence atleast about 90% identical to that set forth in SEQ ID NOs: 159 or 162,and a CDRL3 comprising an amino acid sequence at least about 90%identical to that set forth in SEQ ID NOs: 160 or
 163. 3. A method oftreating cancer comprising administering the antibody or antibodyfragment of claim
 2. 4. A method of treating a viral infectioncomprising administering the antibody or antibody fragment of claim 2.5. A nucleic acid library comprising: a plurality of sequencescomprising nucleic acids that when translated encode for an antibody orantibody fragment, wherein each sequence of the plurality of sequencescomprises a variant sequence encoding for a CDR1, CDR2, or CDR3 on avariable region of a heavy chain (VH) or a CDR1, CDR2, or CDR3 on avariable region of a light chain (VL); wherein the library comprises atleast 30,000 variant sequences; and wherein the antibody or antibodyfragments bind to its antigen with a K_(D) of less than 100 nM.
 6. Thenucleic acid library of claim 5, wherein the antibody is a single domainantibody.
 7. The nucleic acid library of claim 6, wherein the singledomain antibody is a VHH antibody.
 8. The nucleic acid library of claim5, wherein the antibody binds to TIGIT.
 9. The nucleic acid library ofclaim 5, wherein the variable region of the heavy chain when translatedcomprises an amino acid sequence at least about 90% identical to thatset forth in SEQ ID NOs: 84-100.
 10. The nucleic acid library of claim5, wherein the variable region of the light chain when translatedcomprises an amino acid sequence at least about 90% identical to thatset forth in SEQ ID NOs: 101-117.
 11. The nucleic acid library of claim5, wherein the CDR1, CDR2, or CDR3 on the variable region of the heavychain comprises an amino acid sequence at least about 90% identical tothat set forth in any one of SEQ ID NOs: 67-83 or 118-128.
 12. Thenucleic acid library of claim 5, wherein the CDR1, CDR2, or CDR3 on thevariable region of the light chain comprises an amino acid sequence atleast about 90% identical to that set forth in any one of SEQ ID NOs:129-137.
 13. The nucleic acid library of claim 5, wherein the antibodybinds to CD47.
 14. The nucleic acid library of claim 5, wherein theantibody binds to CD3 epsilon.
 15. The nucleic acid library of claim 5,wherein the variable region of the heavy chain when translated comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NOs: 138-141.
 16. The nucleic acid library of claim 5, whereinthe variable region of the light chain when translated comprises anamino acid sequence at least about 90% identical to that set forth inSEQ ID NOs: 142-145.
 17. The nucleic acid library of claim 5, whereinthe nucleic acid library comprises at least 50,000 variant sequences.18. (canceled)
 19. (canceled)
 20. (canceled)
 21. A nucleic acid librarycomprising: a plurality of sequences comprising nucleic acids that whentranslated encode for a single domain antibody, wherein each sequence ofthe plurality of sequences comprises a variant sequence encoding forCDR1, CDR2, or CDR3 on a variable region of a heavy chain (VH); whereinthe library comprises at least 30,000 variant sequences; and wherein theantibody or antibody fragments bind to its antigen with a K_(D) of lessthan 100 nM.
 22. The nucleic acid library of claim 21, wherein a lengthof the VH when translated is about 90 to about 100 amino acids.
 23. Thenucleic acid library of claim 21, wherein a length of the VH whentranslated is about 100 to about 400 amino acids. 24.-51. (canceled)